WorldWideScience

Sample records for human cloned embryo

  1. The First Human Cloned Embryo.

    Science.gov (United States)

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  2. Human embryo cloning prohibited in Hong Kong.

    Science.gov (United States)

    Liu, Athena

    2005-12-01

    Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

  3. The ethics of cloning and human embryo research.

    Science.gov (United States)

    Saran, Madeleine

    2002-01-01

    The successful cloning experiments that led to Dolly in 1997 have raised many ethical and policy questions. This paper will focus on cloning research in human embryonic cells. The possible gains of the research will be judged against the moral issues of doing research on a person. This paper concludes that while the embryo has some moral status, its moral status is outweighed by the multitude of benefits that embryonic stem cell research will bring to humanity. Policy suggestions are given for dealing with this new and developing field of stem cell research.

  4. ES cells derived from cloned embryos in monkey - a jump toward human therapeutic cloning

    Institute of Scientific and Technical Information of China (English)

    Xiangzhong Yang; Sadie L Smith

    2007-01-01

    @@ Therapeutic cloning refers to the derivation of embryonic stem cells (ntESC) from embryos derived from somatic cell nuclear transfer (SCNT) also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus (DNA) removed.

  5. Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    JI Jingjuan; GUO Tonghang; TONG Xianhong; LUO Lihua; ZHOU Guixiang; FU Yingyun; LIU Yusheng

    2007-01-01

    Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P<0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.

  6. Human cloning and embryo research: the 2003 John J. Conley Lecture on medical ethics.

    Science.gov (United States)

    George, Robert P

    2004-01-01

    The author, a member of the U.S. President's Council on Bioethics, discusses ethical issues raised by human cloning, whether for purposes of bringing babies to birth or for research purposes. He first argues that every cloned human embryo is a new, distinct, and enduring organism, belonging to the species Homo sapiens, and directing its own development toward maturity. He then distinguishes between two types of capacities belonging to individual organisms belonging to this species, an immediately exerciseable capacity and a basic natural capacity that develops over time. He argues that it is the second type of capacity that is the ground for full moral respect, and that this capacity (and its concomitant degree of respect) belongs to cloned human embryos no less than to adult human beings. He then considers and rejects counter-arguments to his position, including the suggestion that the capacity of embryos is equivalent to the capacity of somatic cells, that full human rights are afforded only to human organisms with functioning brains, that the possibility of twinning diminishes the moral status of embryos, that the fact that people do not typically mourn the loss of early embryos implies that they have a diminished moral status, that the fact that early spontaneous abortions occur frequently diminishes the moral status of embryos, and that his arguments depend upon a concept of ensoulment. He concludes that if the moral status of cloned human embryos is equivalent to that of adults, then public policy should be based upon this assumption.

  7. [Triploid cloned human embryos: ethical, social, and legal aspects].

    Science.gov (United States)

    Bellver Capella, Vicente

    2012-01-01

    This work attempts to place the experiment within the scientific and social framework of pluripotent-stem-cell research and offer reflections of an ethical and (to a lesser extent) legal nature on the results obtained by this research group. To these ends, the work is divided into two parts. The first part describes the most important aspects of Noggle and Egli's announcement and the biotechnological and media context in which it was made. The second part is concerned with the bioethical issues raised by the experiment. There are basically four issues, which relate to: (1) the nuclear transfer technique, (2) the use of human ovules to carry out the experiment, (3) the destruction of human blastocysts, and (4) the ethical requirements of scientific publications.

  8. Ethical consideration of experimentation using living human embryos: the Catholic Church's position on human embryonic stem cell research and human cloning.

    Science.gov (United States)

    Ohara, N

    2003-01-01

    Although the potential applications of human embryonic stem cells and therapeutic cloning hold promise for the alleged medical benefits, these technologies have posed profound ethical issues because they necessitate the destruction of human embryos. A fundamental point in the issues is the concept of the moral status of human embryos. The Catholic Church has held that human life begins at the moment of conception and therefore, has defended the dignity, inviolable right to life and integrity of human embryos. The Catholic Church has opposed human embryonic stem cell research and any kind of human cloning because they are contrary to the dignity of procreation, of conjugal union and of human embryos. Moreover, these techniques have the risk of creating a sub-category of human beings that are destined basically for the convenience of others. In conclusion, science and technology can never be independent of the criterion of morality, since technology exists for man and must respect his finality.

  9. Statement on Human Cloning

    Science.gov (United States)

    ... ban on efforts to implant a human cloned embryo for the purpose of reproduction. The scientific evidence ... stem cell research, including the use of nuclear transplantation techniques (also known as research or therapeutic cloning), ...

  10. Aberrant DNA methylation in cloned ovine embryos

    Institute of Scientific and Technical Information of China (English)

    LIU Lei; HOU Jian; LEI TingHua; BAI JiaHua; GUAN Hong; AN XiaoRong

    2008-01-01

    By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of cloned ovine embryos. The em-bryos derived from in vitro fertilization were also examined for reference purpose. The results showed that: (1) during the pre-implantation development, cloned embryos displayed a similar demethylation profile to the fertilized embryos; that is, the methylation level decreased to the lowest at 8-cell stage, and then increased again at morulae stage. However, methylation level was obviously higher in cloned embryos than in stage-matched fertilized embryos, especially at 8-cell stage and afterwards; (2) at blastocyst stage, the methylation pattern in cloned embryos was different from that in fertilized em-bryos. In cloned blastocyst, inner cell mass (ICM) exhibited a comparable level to trophectoderm cells (TE), while in in-vitro fertilized blastocyst the methylation level of ICM was lower than that of TE, which is not consistent with that reported by other authors. These results indicate that DNA methylation is abnormally reprogrammed in cloned embryos, implying that aberrant DNA methylation reprogramming may be one of the factors causing cloned embryos developmental failure.

  11. Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    Qiuyan Li; Hengxi Wei; Ying Guo; Yan Li; Rui Zhao; Yufang Ma; Zhengquan Yu; Bo Tang; Lei Zhang; Yunping Dai; Ning Li

    2009-01-01

    Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, s1w3 and s1w6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after acti-vation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.

  12. [Scientific ethics of human cloning].

    Science.gov (United States)

    Valenzuela, Carlos Y

    2005-01-01

    True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

  13. The science, fiction, and reality of embryo cloning.

    Science.gov (United States)

    Cohen, Jacques; Tomkin, Giles

    1994-09-01

    Although many scientists view cloning as a useful procedure for scientific research into early embryo development -- one that cannot currently be used to produce multiple copies of humans -- the popular literature has led some individuals to view it as sinister. To address the concerns of the public, various conceptions of cloning are distinguished and their basis in fact analyzed. The possible uses, benefits, and detriments of both embryo splitting and nuclear transplantation are explained. Once the nature and purposes of cloning are understood, and the distinctive ethical dilemmas created by embryo splitting and nuclear transplantation are sorted out, these procedures should be clinically implemented to assist in vitro fertilization treatment for those who are infertile and to further other therapeutic and investigational efforts in medicine.

  14. Human Cloning

    Science.gov (United States)

    2006-07-20

    genes , for example, has led to new treatments developed by the biotechnology industry for diseases such as diabetes and hemophilia . In the context of...stem cells should be permitted because of the potential for developing new therapies and advancing biomedical knowledge. On May 24, 2005, the House...to describe many different processes that involve making copies of biological material, such as a gene , a cell, a plant or an animal. The cloning of

  15. Islamic perspectives on human cloning.

    Science.gov (United States)

    Sadeghi, Mahmoud

    2007-01-01

    The present paper seeks to assess various views from Islamic jurists relating to human cloning, which is one of the controversial topics in the recent past. Taking Islamic jurisprudence principles, such as the rule of necessity for self preservation and respect for human beings, the rule of la darar wa la dirar ('the necessity to refrain from causing harm to oneself and others') and the rule of usr wa haraj, one may indicate that if human cloning could not be prohibited, as such, it could still be opposed because it gives way to various harmful consequences, which include family disorder, chaos in the clone's family relationships, physical and mental diseases for clones and suffering of egg donors and surrogate mothers. However with due attention to the fact that the reasons behind the prohibition of abortion only restrict the destruction of human embryos in their post-implantation stages, human cloning for biomedical research and exploitation of stem cells from cloned embryos at the blastocyst stage for therapeutic purposes would be acceptable.

  16. The DNA methylation events in normal and cloned rabbit embryos

    Institute of Scientific and Technical Information of China (English)

    TaoChen; Yan-LingZhang; YanJiang; Shu-ZhenLiu; HeideSchatten; Da-YuanChen; Qing-YuanSun

    2005-01-01

    To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite seqnence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and preco-ciously de novo methylated, while the satellite sequence mainrained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellitesequences may have aberrant methylation patterns in cloned embryos.

  17. Market share for semen and cloned embryos in dairy herds.

    NARCIS (Netherlands)

    Boer, de I.J.M.; Arendonk, van J.A.M.

    1994-01-01

    Use of cloned embryos from desirable genotypes (commercial clone lines) enables faster dissemination of superior genetics to dauy producers. Under optimal purchasing strategies of milk producers, the annual proportion of replacement cows from commercial clone lines indicates the market share of clon

  18. Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

    Science.gov (United States)

    Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

    2017-03-01

    Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non-human

  19. Role of glucose in cloned mouse embryo development

    National Research Council Canada - National Science Library

    Zhiming Han; Rita Vassena; Maggie M. Y. Chi; Santhi Potireddy; Miriam Sutovsky; Kelle H. Moley; Peter Sutovsky; Keith E. Latham

    2008-01-01

    Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early...

  20. Development in vitro and mitochondrial fate of interspecies cloned embryos.

    Science.gov (United States)

    Ma, L-B; Yang, L; Hua, S; Cao, J-W; Li, J-X; Zhang, Y

    2008-06-01

    Although the technique of interspecies somatic cell nuclear transfer can be used to increase the population size of endangered mammals, the mitochondrial heteroplasmy in cloned embryos and animals makes this idea doubtful. In present study, goat-sheep cloned embryos were constructed by fusing goat foetal fibroblasts (GFFs) into sheep oocytes and then cultured in vitro to investigate the capability of sheep oocyte dedifferentiating GFF nucleus. Moreover, at each stage of 1- (immediately after fused), 2-, 4-, 8-, 16-cell, morula and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined using real-time PCR. The results showed that: 7.4% of the fused cloned embryos can develop to the blastocyst stage; in the process of one cell to the morula stage, the copy number of two kinds of mtDNA was stable relatively; however, in the process of morula to the blastocyst stage, the decreasing in the copy number of GFF-derived mtDNA, while the increasing in sheep oocyte-derived, resulted in their ratio of decreasing sharply from 2.0 +/- 1.0% to 0.012 +/- 0.004%. This study demonstrates that: (i) the goat-sheep cloned embryos have the ability to develop to blastocyst in vitro; (ii) from the morula stage to the blastocyst stage of goat-sheep cloned embryos, goat derived mitochondria can be gradually replaced with those from sheep oocyte.

  1. BIOETHICS AND HUMAN CLONING

    Directory of Open Access Journals (Sweden)

    Željko Kaluđerović

    2011-12-01

    Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.

  2. Cloning in cattle: from embryo splitting to somatic nuclear transfer.

    Science.gov (United States)

    Heyman, Y; Vignon, X; Chesné, P; Le Bourhis, D; Marchal, J; Renard, J P

    1998-01-01

    The ability to obtain genetically identical offspring in cattle (clones) is useful for research and for potential applications to breeding schemes. Experimental possibilities for generating such animals have evolved considerably in the last two decades. Embryo splitting has become a relatively simple technique but is limited to twinning. Embryonic nuclear transfer has improved and is associated with sexing to generate sets of clones despite a great variability of results between parent embryos. The factors of progress are reviewed here. Recently, somatic cells used as a source of nuclei in bovine nuclear transfer has been demonstrated. Here we present the results of the developmental potential of nuclei from skin and muscle cells.

  3. cDNA cloning, characterization and expression analysis of DTX2, a human WWE and RING-finger gene, in human embryos.

    Science.gov (United States)

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

    The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions.

  4. Should we clone human beings? Cloning as a source of tissue for transplantation.

    Science.gov (United States)

    Savulescu, J

    1999-01-01

    The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910

  5. Should we clone human beings? Cloning as a source of tissue for transplantation.

    Science.gov (United States)

    Savulescu, J

    1999-04-01

    The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus.

  6. Embryos, Clones, and Stem Cells: A Scientific Primer

    Directory of Open Access Journals (Sweden)

    Kenyon S. Tweedell

    2004-01-01

    Full Text Available This article is intended to give the nonspecialist an insight into the nuances of “clones”, cloning, and stem cells. It distinguishes embryonic and adult stem cells, their normal function in the organism, their origin, and how they are recovered to produce stem cell lines in culture. As background, the fundamental processes of embryo development are reviewed and defined, since the manipulation of stem cell lines into desired specialized cells employs many of the same events. Stem cells are defined and characterized and shown how they function in the intact organism during early development and later during cell regeneration in the adult. The complexity of stem cell recovery and their manipulation into specific cells and tissue is illustrated by reviewing current experimentation on both embryonic and adult stem cells in animals and limited research on human stem cell lines. The current and projected use of stem cells for human diseases and repair, along with the expanding methodology for the recovery of human embryonic stem cells, is described. An assessment on the use of human embryonic stem cells is considered from ethical, legal, religious, and political viewpoints.

  7. Reproductive semi-cloning respecting biparental embryo origin: embryos from syngamy between a gamete and a haploidized somatic cell.

    Science.gov (United States)

    Tesarik, J

    2002-08-01

    Embryos formed by somatic cell nuclear transfer to enucleated oocytes (cloning) have given rise to viable offspring in several mammalian species. The possibility of future application of this technique to human assisted reproduction (reproductive cloning) has been widely debated. On this background there is current discussion of the potential for a cloning-derived technique, which aims at syngamy between a gamete nucleus from one parent and a somatic cell nucleus from the other. Critical analysis of the clinical indications, the current state of the art, biological concerns and ethical considerations relative to this technique, called here reproductive semi-cloning, are presented. Such a technique requires validation by further research before it can be considered as a treatment option. This debate explores issues raised by the technique.

  8. Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed embryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.

  9. [The human embryo after Dolly: new practices for new times].

    Science.gov (United States)

    de Miguel Beriain, Iñigo

    2008-01-01

    The possiblity of cloning human beings introduced a lot of issues in our ethical and legal frameworks. In this paper, we will put the focus into the necessary changes in the concept of embryo that our legal systems will have to implement in order to face the new situation. The description of the embryo as a group of cells able to develop into a human being will be defended here as the best way of doing so.

  10. Employing mated females as recipients for transfer of cloned dog embryos.

    Science.gov (United States)

    Kim, Geon A; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Park, Eun Jung; Lim, Sang Hyun; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

    2013-01-01

    It has been suggested that co-transferring parthenogenetic embryos could improve the pregnancy success rate with cloned embryos in mammals. As an alternative to co-transferring parthenotes, in dogs we employed recipient females that possessed in vivo-fertilised embryos as a result of mating to determine whether mated bitches could be suitable recipients for cloned embryos. The effect of using mated recipients on implantation and pregnancy rates of canine somatic cell nuclear transfer embryos was also determined. Cloned embryos were transferred into the oviducts of naturally synchronous females that had mated with male dogs before ovulation. The pregnancy rate appeared to be similar between mated recipients (50%) and non-mated recipients (28.57%; P>0.05). However, the delivery rate of cloned pups was significantly higher in mated recipients than non-mated recipients (10.53 vs 2.38%; Pdogs can be used as recipients for cloned embryos.

  11. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  12. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

    Directory of Open Access Journals (Sweden)

    Byungkuk Min

    2016-05-01

    Full Text Available Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.

  13. Influence of embryo handling and transfer method on pig cloning efficiency.

    Science.gov (United States)

    Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

    2015-03-01

    The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs.

  14. Procreative liberty, enhancement and commodification in the human cloning debate.

    Science.gov (United States)

    Shapshay, Sandra

    2012-12-01

    The aim of this paper is to scrutinize a contemporary standoff in the American debate over the moral permissibility of human reproductive cloning in its prospective use as a eugenic enhancement technology. I shall argue that there is some significant and under-appreciated common ground between the defenders and opponents of human cloning. Champions of the moral and legal permissibility of cloning support the technology based on the right to procreative liberty provided it were to become as safe as in vitro fertilization and that it be used only by adults who seek to rear their clone children. However, even champions of procreative liberty oppose the commodification of cloned embryos, and, by extension, the resulting commodification of the cloned children who would be produced via such embryos. I suggest that a Kantian moral argument against the use of cloning as an enhancement technology can be shown to be already implicitly accepted to some extent by champions of procreative liberty on the matter of commodification of cloned embryos. It is in this argument against commodification that the most vocal critics of cloning such as Leon Kass and defenders of cloning such as John Robertson can find greater common ground. Thus, I endeavor to advance the debate by revealing a greater degree of moral agreement on some fundamental premises than hitherto recognized.

  15. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    Science.gov (United States)

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  16. Cell phoney: human cloning after Quintavalle.

    Science.gov (United States)

    Morgan, Derek; Ford, Mary

    2004-12-01

    Reproductive cloning has thrown up new scientific possibilities, ethical conundrums, and legal challenges. An initial question, considered by the English courts in 2003, was whether the technique presently available, that of cell nucleus replacement, falls outside the provisions of the Human Fertilisation and Embryology Act 1990. If it does, the creation and use, including use in research protocols, of human embryos would be unregulated, disclosing a need to consider remedial legislation. The resolution by the courts of this legal question dramatically engages them in a resolution of fundamental ethical dilemmas, and discloses the possibilities and limitation of negotiating science policy through the processes of litigation.

  17. Equine cloning: in vitro and in vivo development of aggregated embryos.

    Science.gov (United States)

    Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

    2012-07-01

    The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

  18. Statement on Human Cloning

    Science.gov (United States)

    ... as our understanding of this technology advances. Support Stem Cell Research (including Research Cloning) AAAS supports stem cell research, including the use of nuclear transplantation techniques (also ...

  19. Human cloning and child welfare.

    Science.gov (United States)

    Burley, J; Harris, J

    1999-01-01

    In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

  20. Human cloning and 'posthuman' society.

    Science.gov (United States)

    Blackford, Russell

    2005-01-01

    Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning.

  1. HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yingying [Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223 (China); State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Hai, Tang; Liu, Zichuan; Zhou, Shuya; Lv, Zhuo [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Ding, Chenhui; Liu, Lei [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Niu, Yuyu [Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223 (China); Zhao, Xiaoyang; Tong, Man [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Liu [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China); Jouneau, Alice [INRA, UMR 1198, ENVA, CNRS, FRE 2857, Biologie du Developpement et Reproduction, Jouy en Josas F-78350 (France); Zhang, Xun [Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 (United States); Ji, Weizhi, E-mail: wji@mail.kiz.ac.cn [Department of Reproduction and Development, Kunming Institute of Zoology and Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223 (China); Zhou, Qi, E-mail: qzhou@ioz.ac.cn [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080 (China)

    2010-07-02

    Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.

  2. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  3. Development of interspecies cloned embryos in yak and dog.

    Science.gov (United States)

    Murakami, Masao; Otoi, Takeshige; Wongsrikeao, Pimprapar; Agung, Budiyanto; Sambuu, Rentsenkhand; Suzuki, Tatsuyuki

    2005-01-01

    Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.

  4. Reconstruction of human embryos derived from somatic cells

    Institute of Scientific and Technical Information of China (English)

    LU Changfu; LIN Ge; XIE Changqing; GONG Fei; ZHOU Hong; TAN Yueqiu; LU Guangxiu

    2003-01-01

    Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into MⅡ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyte activation and 2PN formation, we removed the female PN. By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation, and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into theblastocyst stage in vitro.

  5. Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

    Science.gov (United States)

    Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

    2009-09-01

    Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

  6. Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

    National Research Council Canada - National Science Library

    Chung, Young Gie; Mann, Mellissa R W; Bartolomei, Marisa S; Latham, Keith E

    2002-01-01

    .... Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined...

  7. Nuclear-Cytoplasmic “Tug of War” During Cloning: Effects of Somatic Cell Nuclei on Culture Medium Preferences of Preimplantation Cloned Mouse Embryos1

    National Research Council Canada - National Science Library

    Young Gie Chung; Mellissa R. W. Mann; Marisa S. Bartolomei; Keith E. Latham

    2002-01-01

    .... Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined...

  8. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

  9. The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

    Science.gov (United States)

    Rim, Chol Ho; Fu, Zhixin; Bao, Lei; Chen, Haide; Zhang, Dan; Luo, Qiong; Ri, Hak Chol; Huang, Hefeng; Luan, Zhidong; Zhang, Yan; Cui, Chun; Xiao, Lei; Jong, Ui Myong

    2013-12-01

    To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, pcloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

  10. In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

    Directory of Open Access Journals (Sweden)

    Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

    2014-02-01

    Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

  11. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  12. Towards an understanding of British public attitudes concerning human cloning.

    Science.gov (United States)

    Shepherd, Richard; Barnett, Julie; Cooper, Helen; Coyle, Adrian; Moran-Ellis, Jo; Senior, Victoria; Walton, Chris

    2007-07-01

    The ability of scientists to apply cloning technology to humans has provoked public discussion and media coverage. The present paper reports on a series of studies examining public attitudes to human cloning in the UK, bringing together a range of quantitative and qualitative methods to address this question. These included a nationally representative survey, an experimental vignette study, focus groups and analyses of media coverage. Overall the research presents a complex picture of attitude to and constructions of human cloning. In all of the analyses, therapeutic cloning was viewed more favourably than reproductive cloning. However, while participants in the focus groups were generally negative about both forms of cloning, and this was also reflected in the media analyses, quantitative results showed more positive responses. In the quantitative research, therapeutic cloning was generally accepted when the benefits of such procedures were clear, and although reproductive cloning was less accepted there was still substantial support. Participants in the focus groups only differentiated between therapeutic and reproductive cloning after the issue of therapeutic cloning was explicitly raised; initially they saw cloning as being reproductive cloning and saw no real benefits. Attitudes were shown to be associated with underlying values associated with scientific progress rather than with age, gender or education, and although there were a few differences in the quantitative data based on religious affiliation, these tended to be small effects. Likewise in the focus groups there was little direct appeal to religion, but the main themes were 'interfering with nature' and the 'status of the embryo', with the latter being used more effectively to try to close down further discussion. In general there was a close correspondence between the media analysis and focus group responses, possibly demonstrating the importance of media as a resource, or that the media reflect

  13. Superovulation of the Cloned Cattle Derived from Somatic Cells and the Transfer of the Vitrified-Thawed Embryos of the Cloning Cattle

    Institute of Scientific and Technical Information of China (English)

    DONG Ya-juan; BAI Xue-jin; LI Jian-dong; CHENG Ming

    2004-01-01

    In this experiment, it was designed to carry out superovulation on the two cloned cattles, vitrification and transfer of the embryos recovered from them. First of all, it was carried out vitrification on embryos obtained by IVF. Results showed that there were no significant differences between the blastocysts (obtained by IVF) vitrified in EPS10 and these in EPS20 on the resuscitative rate and the developmental rate. The hatched rate of the blastocysts vitrified in EPS10 (31.3%, 35/112) was significantly higher than that in EPS20 (12.2%, 13/107) (P<0.01), so EPS20 was selected as the vitrification solution to freeze the embryos recovered from the cloned cattle. After superovulation, six (four usable embryos) and ten (nine usable embryos) embryos were respectively recovered from Kangkang and Shuanghuang. Two embryos were selected from the recovered embryos of each cloned cattle to freeze in EPS20, subsequently thawed and transferred into luteal ipsilateral uterine horns of 4 Holstein recipient cows after synchronization of estrus, respectively. At last, one recipient cow (No. 9908) became pregnant and delivered one healthy calf (descendant of the cloned cattle-Shuangshuang). The results of this experiment show that the cloned cattle as well as common cattle had better response to the exotic FSH and better ability to multiovulation, the embryos recovered from the cloned cattle can be vitrificated.

  14. Human cloning, stem cell research. An Islamic perspective.

    Science.gov (United States)

    Al-Aqeel, Aida I

    2009-12-01

    The rapidly changing technologies that involve human subjects raise complex ethical, legal, social, and religious issues. Recent advances in the field of cloning and stem cell research have introduced new hopes for the treatment of serious diseases. But this promise has raised many complex questions. This field causes debate and challenge, not only among scientists but also among ethicists, religious scholars, governments, and politicians. There is no consensus on the morality of human cloning, even within specific religious traditions. In countries in which religion has a strong influence on political decision making, the moral status of the human embryo is at the center of the debate. Because of the inevitable consequences of reproductive cloning, it is prohibited in Islam. However, stem cell research for therapeutic purposes is permissible with full consideration, and all possible precautions in the pre-ensoulment stages of early fetus development, if the source is legitimate.

  15. Human embryos in the original position?

    Science.gov (United States)

    DiSilvestro, Russell

    2005-06-01

    Two different discussions in John Rawls' A Theory of Justice lead naturally to a rather conservative position on the moral status of the human embryo. When discussing paternalism, he claims that the parties in the original position would seek to protect themselves in case they end up as incapacitated or undeveloped human beings when the veil of ignorance is lifted. Since human embryos are examples of such beings, the parties in the original position would seek to protect themselves from their embryonic stages onward. When discussing the basis of equality, Rawls claims that the parties in the original position would guarantee basic rights for all those with the capacity to take part in this original position. To guarantee the basic rights of infants and young children, he goes on to interpret this capacity as a "potentiality that is ordinarily realized in due course." Since human embryos have this potentiality, they too should have basic rights.

  16. Human reproductive cloning: a conflict of liberties.

    Science.gov (United States)

    Havstad, Joyce C

    2010-02-01

    Proponents of human reproductive cloning do not dispute that cloning may lead to violations of clones' right to self-determination, or that these violations could cause psychological harms. But they proceed with their endorsement of human reproductive cloning by dismissing these psychological harms, mainly in two ways. The first tactic is to point out that to commit the genetic fallacy is indeed a mistake; the second is to invoke Parfit's non-identity problem. The argument of this paper is that neither approach succeeds in removing our moral responsibility to consider and to prevent psychological harms to cloned individuals. In fact, the same commitment to personal liberty that generates the right to reproduce by means of cloning also creates the need to limit that right appropriately. Discussion of human reproductive cloning ought to involve a careful and balanced consideration of both the relevant aspects of personal liberty - the parents' right to reproductive freedom and the cloned child's right to self-determination.

  17. Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

    Directory of Open Access Journals (Sweden)

    Wang Ya-Ping

    2008-03-01

    Full Text Available Abstract Background Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp in length, with a 342 bp open reading frame (ORF encoding a putative protein of 113 amino acids (aa. Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

  18. Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

  19. Scientists Create Part-Human, Part-Pig Embryo

    Science.gov (United States)

    ... 163262.html Scientists Create Part-Human, Part-Pig Embryo One goal of this stem cell research is ... have successfully used human stem cells to create embryos that are part-human, part-pig. Scientists said ...

  20. Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos

    DEFF Research Database (Denmark)

    Li, J; Villemoes, K; Zhang, Y

    2009-01-01

    The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41â€"42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade...

  1. Reproductive cloning in humans and therapeutic cloning in primates: is the ethical debate catching up with the recent scientific advances?

    Science.gov (United States)

    Camporesi, S; Bortolotti, L

    2008-09-01

    After years of failure, in November 2007 primate embryonic stem cells were derived by somatic cellular nuclear transfer, also known as therapeutic cloning. The first embryo transfer for human reproductive cloning purposes was also attempted in 2006, albeit with negative results. These two events force us to think carefully about the possibility of human cloning which is now much closer to becoming a reality. In this paper we tackle this issue from two sides, first summarising what scientists have achieved so far, then discussing some of the ethical arguments in favour and against human cloning which are debated in the context of policy making and public consultation. Therapeutic cloning as a means to improve and save lives has uncontroversial moral value. As to human reproductive cloning, we consider and assess some common objections and failing to see them as conclusive. We do recognise, though, that there will be problems at the level of policy and regulation that might either impair the implementation of human reproductive cloning or make its accessibility restricted in a way that could become difficult to justify on moral grounds. We suggest using the time still available before human reproductive cloning is attempted successfully to create policies and institutions that can offer clear directives on its legitimate applications on the basis of solid arguments, coherent moral principles, and extensive public consultation.

  2. Cloning humans? Current science, current views, and a perspective from Christianity.

    Science.gov (United States)

    Brun, Rudolf B

    2002-01-01

    Therapeutic cloning is urgent and should be vigorously supported. To successfully argue for this position, the distinction between a human embryo and a human nuclear transplant may be helpful. Even if current technical difficulties should be solved, global legislation should prohibit cloning for the purpose of fabricating babies. This position originates from a view on human nature in general and from a Christian perspective in particular.

  3. Effects of embryo-derived exosomes on the development of bovine cloned embryos

    Science.gov (United States)

    Qu, Pengxiang; Qing, Suzhu; Liu, Ruiqi; Qin, Hongyu; Wang, Weiwei; Qiao, Fang; Ge, Hui; Liu, Jun; Zhang, Yong; Wang, Yongsheng

    2017-01-01

    The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation. PMID:28350875

  4. Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos.

    Science.gov (United States)

    Park, Mi-Ryung; Im, Gi-Sun; Kim, Sung Woo; Hwang, Seongsoo; Park, Jae-Hong; Kim, Hyun; Do, Yoon Jung; Park, Soo Bon; Yang, Bo-Suck; Song, Young Min; Cho, Jae-Hyeon; Ko, Yeoung-Gyu

    2013-06-01

    The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

  5. Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology.

    Science.gov (United States)

    Skrzyszowska, Maria; Karasiewicz, Jolanta; Bednarczyk, Marek; Samiec, Marcin; Smorag, Zdzisław; Waś, Bogusław; Guszkiewicz, Andrzej; Korwin-Kossakowski, Maciej; Górniewska, Maria; Szablisty, Ewa; Modliński, Jacek A; Łakota, Paweł; Wawrzyńska, Magdalena; Sechman, Andrzej; Wojtysiak, Dorota; Hrabia, Anna; Mika, Maria; Lisowski, Mirosław; Czekalski, Przemysław; Rzasa, Janusz; Kapkowska, Ewa

    2006-01-01

    The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds

  6. Human embryo twinning with applications in reproductive medicine.

    Science.gov (United States)

    Illmensee, Karl; Levanduski, Mike; Vidali, Andrea; Husami, Nabil; Goudas, Vasilios T

    2010-02-01

    To assess the efficacy of human embryo twinning by blastomere biopsy at different early embryonic stages (splitting efficiency) and to determine the in vitro developmental capacity of twinned human embryos (developmental efficiency). Randomized comparative study. Private IVF centers. Couples undergoing IVF donating triploid embryos. Embryos at the 2- to 5- and 6- to 8-cell stage were split into twin embryos. Half the number of blastomeres from donor embryos were removed and inserted into recipient empty zonae pellucidae. After embryo splitting, donor and recipient embryos were cultured in vitro. Development of twinned embryos to the blastocyst stage. The number of developing embryos obtained after splitting could be increased in comparison with the number of embryos available before splitting at the 6- to 8-cell stage but not at the 2- to 5-cell stage (splitting efficiency). Splitting of 6- to 8-cell embryos yielded superior rates of twin embryos developing to blastocysts (developmental efficiency). Twinning success was related to the superior morphological quality of embryos used for splitting. This is the first report on twinned human embryos developing to blastocysts. This study exhibits the potential for novel applications in human assisted reproduction. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  7. "Goodbye Dolly?" The ethics of human cloning.

    Science.gov (United States)

    Harris, J

    1997-01-01

    The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity. PMID:9451604

  8. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.

    Science.gov (United States)

    Eggan, K; Akutsu, H; Loring, J; Jackson-Grusby, L; Klemm, M; Rideout, W M; Yanagimachi, R; Jaenisch, R

    2001-05-22

    To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

  9. Effect of roscovitine-treated donor cells on development of porcine cloned embryos.

    Science.gov (United States)

    Park, H J; Koo, O J; Kwon, D K; Kang, J T; Jang, G; Lee, B C

    2010-12-01

    Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 μM roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production. © 2009 Blackwell Verlag GmbH.

  10. Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis.

    Science.gov (United States)

    Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing

    2013-03-15

    Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality.

  11. Birth of cloned calves from vitrified-warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning.

    Science.gov (United States)

    Saha, Ambikaprasanna; Panda, Sudeepta K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K

    2013-01-01

    The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2±2.2% vs 41.7±1.7% and 39.1±2.1%, respectively; Pcloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

  12. Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

    Science.gov (United States)

    Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

    2013-10-01

    The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed.

  13. In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

    DEFF Research Database (Denmark)

    Holm, P; Nagashima, H; Sun, F-J

    1995-01-01

    Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development...... of cloned embryos were compared using: l. In vivo oocytes and in vivo culture; 2. In vivo oocytes and in vitro culture; and 3. In vitro oocytes and in vitro culture. Selected embryos were transferred to recipients. Donor embryos and oviductal oocytes were collected from superovulated Merino ewes. In vivo...... matured oocytes were enucleated and fused with inserted blastomeres from donor embryos. In vitro matured oocytes were enucleated and allowed to age prior to blastomere insertion and electrofusion. Fused embryos were cultured for approximately 132 h either in vivo in ligated sheep oviducts or in vitro...

  14. The ethics of human reproductive cloning.

    Science.gov (United States)

    Strong, Carson

    2005-03-01

    This article addresses the question of whether human reproductive cloning could be ethically justifiable in at least some cases involving infertile couples who would choose cloning as a way to have a genetically related child. At present, the risk of congenital anomalies constitutes a compelling argument against human reproductive cloning. The article explores whether reproductive cloning could be ethically justifiable if, at some future time, cloning becomes possible without an elevated risk of anomalies. It is argued that freedom to use cloning is a form of procreative freedom and, as such, deserves respect. All of the objections that have been raised against human reproductive cloning fall under three main categories: those that appeal to the interests of the child, those based on consequences for society, and those arising from teleological views. Objections that appeal to the child's interests are, in turn, of two main kinds: consequentialist and deontological. All of these types of objections are examined, and it is found that each involves serious problems that prevent it from being a reasonable objection in the context of the infertility cases considered. It is concluded that human reproductive cloning would be ethically justifiable in at least some cases involving infertile couples, provided that it could be performed without an elevated risk of anomalies.

  15. Human Cloning: Let's Discuss It.

    Science.gov (United States)

    Taras, Loretta; Stavroulakis, Anthea M.; Ortiz, Mary T.

    1999-01-01

    Describes experiences with holding discussions on cloning at a variety of levels in undergraduate biology courses. Discusses teaching methods used and student reactions to the discussions. Contains 12 references. (WRM)

  16. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    OpenAIRE

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF...

  17. Quantitative analysis of mitochondrial RNA in goat-sheep cloned embryos.

    Science.gov (United States)

    Ma, Li-Bing; Yang, Li; Zhang, Yong; Cao, Jun-Wei; Hua, Song; Li, Ji-Xia

    2008-01-01

    Mitochondria are the key generators of cellular ATP, and contain extranuclear genome-mitochondrial DNA (mtDNA). In the process of nuclear transfer (NT), heteroplasmic sources of mtDNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. Previous studies showed inconsistent patterns of mtDNA inheritance in offspring and early fetuses generated through interspecies NT. The quantitative analysis of mitochondrial RNA (mtRNA) in interspecies cloned embryos is useful for better understanding the fate of two types of mitochondria. The components of nicotinamide adenine dinucleotide (NADH) dehydrogenase were coded by both nuclear DNA (nDNA) and mtDNA. The Subunit 1 (ND-1) is one of seven NADH dehydrogenase subunits coded by mtDNA. In present study, using real-time and reverse-transcription PCR, the copy number of species-specific ND-1 mRNA was examined in goat-sheep cloned embryos of various developmental stages, and was applied to evaluate the expression pattern of species-specific mtDNA. The results of showed that (1) the expression of mtDNA derived from goat fetal fibroblast (GFF) decreased from 1-cell stage (immediately after fused) to 2-cell stage, and could not be detected from 4-cell stage onward to blastocyst stage; (2) the expression of mtDNA derived from sheep oocyte was roughly constant from 1-cell stage to the 8-cell stage, increased gradually from 16-cell stage, and sharply at morula and blastocyst stage. Moreover, we strongly argued a mechanism, that is GFF-derived mitochondria were degraded for the depression of bioenergetic functions, and then selectively eliminated during the embryogenesis of goat-sheep cloned embryos.

  18. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  19. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    Science.gov (United States)

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  20. Cryopreservation of embryos and oocytes in human assisted reproduction.

    Science.gov (United States)

    Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor

    2014-01-01

    Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

  1. Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    János Konc

    2014-01-01

    Full Text Available Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification of human embryos and oocytes are summarized.

  2. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    Directory of Open Access Journals (Sweden)

    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  3. [Human cloning in Muslim and Arab law].

    Science.gov (United States)

    Aldeeb Abu-Sahlieh, Sami A

    2009-01-01

    Cloning is a modern medical procedure that Muslim religious authorities treat en resorting to the general principles established by classical Muslim law based on the Koran and the Sunnah of Muhhamad as the messenger of God. In this regard, human beings are not capable of deciding what is or what is not lawful without resorting to divine norms. Cloning clashes with several principles. Firstly, the principle of the respect for life in relation to surpernumeraries, but Muslim authors are not in unanimous agreement on the determination of the moment at which life begins. Secondly, is the respect of progeny: cloning could only take place between a married couple. But even if these two principles are respected, cloning poses two major problems: the diversity of species expounded by the Koran and the Sunnah and a lack of interest. Which explains the quasi-unanimous opposition of Muslim writings regarding cloning.

  4. Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

    Institute of Scientific and Technical Information of China (English)

    Hengxi Wei; Qiuyan Li; Jun Li; Yan Li; Yunping Dai; Yufang Ma; Kai Xue; Ning Li

    2008-01-01

    Cloning pigs by somatic cell nuclear transfer (SCNT) has wide applications in basic research,human medicine and agricultural production.To improve cloning efficiency,the effect of two basic maturation media,NCSU-23 and TCMI99,was compared,and TCM199 was selected for the following experiments with leptin.We systematically studied the effects of leptin supplementation on oocytes in vitro maturation (IVM),in vitro development of parthenogenetically activated (Phi) and SCNT embryos and/n vivo develop-ment of SCNT embryos after embryo transfer (ET).The results showed that supplementation of 100 or 200 ng/ml leptin into the mat-uration medium did not greatly affect nuclear maturation of oocytes,or cleavage rates of PA and SCNT (P<0.05).Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium,and the number of cells in PA blastocysts was also improved (P<0.05).The number of cells in blastocyst of SCNT was improved,when 100 ng/ml leptin was added (P<0.05).Furthermore,supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate in pig cloning.

  5. Functional analysis of bovine Nramp1 and production of transgenic cloned embryos in vitro.

    Science.gov (United States)

    Cheng, Xiang; Yu, Xiaoli; Liu, Yajun; Deng, Jie; Ma, Xiaoling; Wang, Huayan

    2015-02-01

    Natural resistance-associated macrophage protein 1 (Nramp1) plays an important role in restraining the growth of intracellular pathogens within macrophages. In this study, Nramp1 cDNA was cloned from Qinchuan cattle and its anti-bacterial activity was demonstrated as being able to significantly inhibit the growth of Salmonella abortusovis and Brucella abortus in macrophages. Calf fibroblasts stably transfected with pSP-NRAMP1-HA vector were used to reconstruct bovine embryos by somatic cell nuclear transfer (SCNT). Reconstructed embryos were maturated in vitro and the blastocyst formation rate (14.0%) was similar to that of control embryos (14.5%). Transgenic blastocysts were transplanted into 43 recipient cattle, of which 14 recipients became pregnant as evidenced by non-return estrus and by rectal palpation. One fetus was aborted after 6½ months of pregnancy and transgene integration was confirmed by semi-quantitative polymerase chain reaction. Together, this study showed that bovine Nramp1 retains biological function against the growth of intracellular bacteria and can be used to reconstruct embryos and produce Nramp1 transgenic cattle, which may benefit the animal and enhance their ability to prevent attack by intracellular pathogens.

  6. Emotional reactions to human reproductive cloning.

    Science.gov (United States)

    May, Joshua

    2016-01-01

    Extant surveys of people's attitudes towards human reproductive cloning focus on moral judgements alone, not emotional reactions or sentiments. This is especially important given that some (especially Leon Kass) have argued against such cloning on the ground that it engenders widespread negative emotions, like disgust, that provide a moral guide. To provide some data on emotional reactions to human cloning, with a focus on repugnance, given its prominence in the literature. This brief mixed-method study measures the self-reported attitudes and emotions (positive or negative) towards cloning from a sample of participants in the USA. Most participants condemned cloning as immoral and said it should be illegal. The most commonly reported positive sentiment was by far interest/curiosity. Negative emotions were much more varied, but anxiety was the most common. Only about a third of participants selected disgust or repugnance as something they felt, and an even smaller portion had this emotion come to mind prior to seeing a list of options. Participants felt primarily interested and anxious about human reproductive cloning. They did not primarily feel disgust or repugnance. This provides initial empirical evidence that such a reaction is not appropriately widespread. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  7. Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

    Institute of Scientific and Technical Information of China (English)

    Wang ErYao; YU Yang; Li XueMei; JIAO LiHong; Wang Liu

    2007-01-01

    A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse activation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.

  8. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  9. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  10. Human reproductive cloning and reasons for deprivation.

    Science.gov (United States)

    Jensen, D A

    2008-08-01

    Human reproductive cloning provides the possibility of genetically related children for persons for whom present technologies are ineffective. I argue that the desire for genetically related children is not, by itself, a sufficient reason to engage in human reproductive cloning. I show this by arguing that the value underlying the desire for genetically related children implies a tension between the parent and the future child. This tension stems from an instance of a deprivation and violates a general principle of reasons for deprivation. Alternative considerations, such as a right to procreative autonomy, do not appear helpful in making the case for human reproductive cloning merely on the basis of the desire for genetically related children.

  11. The fate of the mosaic embryo : chromosomal constitution and development of Day 4, 5 and 8 human embryos

    NARCIS (Netherlands)

    Santos, Margarida Avo; Teklenburg, Gijs; Macklon, Nick S.; Van Opstal, Diane; Schuring-Blom, G. Heleen; Krijtenburg, Pieter-Jaap; de Vreeden-Elbertse, Johanna; Fauser, Bart C.; Baart, Esther B.

    2010-01-01

    Post-zygotic chromosome segregation errors are very common in human embryos after in vitro fertilization, resulting in mosaic embryos. However, the significance of mosaicism for the developmental potential of early embryos is unknown. We assessed chromosomal constitution and development of embryos f

  12. The human embryo in the Christian tradition: a reconsideration.

    Science.gov (United States)

    Jones, D A

    2005-12-01

    Recent claims that the Christian tradition justifies destructive research on human embryos have drawn upon an article by the late Professor Gordon Dunstan which appeared in this journal in 1984. Despite its undoubted influence, this article was flawed and seriously misrepresented the tradition of Christian reflection on the moral status of the human embryo.

  13. Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage.

    Science.gov (United States)

    Zhao, Q; Qiu, Y G; Tian, J T; Wang, C S; An, T Z

    2016-11-17

    The cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P M-embryos were octoploid before the first cleavage. Furthermore, 81.25% of the blastomeres of blastocysts developed from the M-embryos showed abnormal ploidy compared with those developed from the G1-embryos (22.55%). However, some of the blastomeres remained diploid in all the M-embryos tested. A portion of the blastomeres restored normal diploidy in some of the M-embryos at the blastocyst stage. This finding provides an explanation for M-embryos developing to term.

  14. The risk of introduction of equine infectious anemia virus into USA via cloned horse embryos imported from Canada.

    Science.gov (United States)

    Asseged, B D; Habtemariam, T; Tameru, B; Nganwa, D

    2012-01-15

    Deriving horse oocytes in the USA is hampered by the lack of abattoirs processing horse carcasses which could provide abundant quantities of ovaries from slaughtered mares. Therefore, several cloning industries in the USA are attempting to import cloned horse embryos from Canada. Like any agricultural commodity, cloned embryos pose a risk of introduction of exotic animal diseases into the importing country. Under such circumstances, risk assessment could provide an objective, transparent, and internationally accepted means for evaluating the risk. This quantitative risk assessment (QRA) was initiated to determine the risk of introduction of Equine infectious anemia virus (EIAV) into the USA via cloned horse embryos imported from Canada. In assessing the risk, a structured knowledge base regarding cloning in relation to Equine infectious anemia (EIA) was first developed. Based on the knowledge base, a scenario tree was developed to determine conditions (with mathematical probabilities) that could lead to the introduction and maintenance of EIAV along the cloning pathway. Parameters for the occurrence of the event at each node were estimated using published literature. Using @Risk software and setting Monte Carlo simulation at 50,000 iterations, the probability of importing an EIAV-infected cloned horse embryo was 1.8 × 10(-9) (R = 1.5 × 10(-12) to 2.9 × 10(-8)). Taking into account the current protocol for equine cloning and assuming the yield of 5 to 30 clones per year, the possible number of EIAV-infected cloned horse embryos ranged from 2.0 × 10(-10) to 9.1 × 10(-5) (Mean = 1.4×10(-6)) per year. Consequently, it would take up to 1.5 × 10(7) (R = 1.6 × 10(4) to 5.1 × 10(10)) years for EIAV to be introduced into the USA. Based on the knowledge base and our critical pathway analysis, the biological plausibility of introducing EIAV into USA via cloned horse embryos imported from Canada is extremely low. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. MOLECULAR CLONING OF HUMAN NEUROTROPHIN-4 GENE

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Cloning and sequencing of the human neurotrophin-4(hNT-4) gene.Methods With the chromosomal DNA of human blood lymphocytes as template,hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger's single stranded DNA terminal termination method.Results The sequence of the cloned gene is completely the same as that reported in the literature(GenBank data base,M86528).Conclusion This study successfully cloning and sequenced the gene of mhNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote,and to continue the research on the gene therapy of Alzheimer's disease intensively.This study indicate that the hNT-4 is conservative in different races and individuals.

  16. Human cloning: three mistakes and an alternative.

    Science.gov (United States)

    Baylis, Françoise

    2002-06-01

    The current debate on the ethics of cloning humans is both uninspired and uninspiring. In large measure this is because of mistakes that permeate the discourse, including the mistake of thinking that cloning technology is strictly a reproductive technology when it is used to create whole beings. As a result, the challenge this technology represents regarding our understanding of ourselves and the species to which we belong typically is inappropriately downplayed or exaggerated. This has meant that important (albeit disquieting) societal issues and species-type concerns have not been fully explored. This paper, intended as a corrective, suggests that we take an alternate view of human cloning as both an enhancement and a reproductive technology. This proposed shift in the framework for analysis counters the current narrow framing of the issues and introduces new questions about the prospect of modifying the species.

  17. Cryopreservation of Oocytes and Embryos in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    Konc J

    2005-01-01

    Full Text Available Cryopreservation has become an integral component of assisted reproductive technology. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals prior to cancer treatments. While the efficiency of oocyte and embryo freezing technology has increased over time, there is still room for improvement, since even under ideal circumstances the clinical pregnancy rate from frozen embryo transfer is approximately two-thirds of that from the fresh transfer of embryos. Thus, studies connected with cryopreservation of human oocytes and embryos are very important to the expansion of effective clinical services. This review gives a summary of the theoretical and technical aspects of oocyte and embryo cryopreservation.

  18. Pregnancies and piglets from large white sow recipients after two transfer methods  of cloned  and trangenic embryos of different pig breeds

    DEFF Research Database (Denmark)

    Schmidt, Mette; Kragh, Peter Michael; Li, Juan;

    2010-01-01

    The aim of this study was to report from a larger study with pregnancy and delivery results after transfer of cloned transgenic/non-transgenic Large White or minipig embryos to Large White sow recipients. The effect of both total numbers of transferred embryos as well as site of their deposition...... (uni- vs. bi-lateral) was studied. Four to five days after natural heat, 85 Large White (LW) sows received Day 5 or 6 handmade cloned embryos. Large White embryos were non-transgenic and were transferred to 36 recipients, while 49 recipients each received Minipig embryos, either non......-transgenic or with 1 of 4 types of transgenes. Furthermore, the number of embryos transferred was in two categories, as 46 recipients received 40-60 embryos while 39 received 60-120 embryos. Finally, in 59 of the recipients embryos were transferred to one of the uterine horns (unicornual) while 26 other recipients had...

  19. Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos.

    Science.gov (United States)

    Popken, Jens; Brero, Alessandro; Koehler, Daniela; Schmid, Volker J; Strauss, Axel; Wuensch, Annegret; Guengoer, Tuna; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

    2014-01-01

    Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.

  20. [Medical, ethical and legal issues in cryopreservation of human embryos].

    Science.gov (United States)

    Beca, Juan Pablo; Lecaros, Alberto; González, Patricio; Sanhueza, Pablo; Mandakovic, Borislava

    2014-07-01

    Embryo cryopreservation improves efficiency and security of assisted reproduction techniques. Nonetheless, it can be questionable, so it must be justified from technical, legal and ethical points of view. This article analyses these perspectives. Embryo cryopreservation maximizes the probability of pregnancy, avoids new ovary stimulations and reduces the occurrence of multiple gestations. There is consensus that the in vitro embryo deserves legal protection by its own, although not as a newborn. Very few countries prohibit embryo cryopreservation based on the legal duty to protect human life since fecundation. Those countries that allow it, privilege women's reproductive rights. In Chile and in Latin America, no laws have been promulgated to regulate human assisted reproduction. The moral status of the embryo depends on how it is considered. Some believe it is a potential person while others think it is just a group of cells, but all recognize that it requires some kind of respect and protection. There is lack of information about the number of frozen embryos and their final destination. As a conclusion the authors propose that women or couples should have the right to decide autonomously, while institutions ought to be clear in their regulations. And the legislation must establish the legal status of the embryo before its implantation, the couples' rights and the regulation of the embryo cryopreservation. Personal, institutional or legal decisions must assume a concept about the moral status of the human embryo and try to avoid their destruction or indefinite storage.

  1. Investigation of DNA repair in human oocytes and preimplantation embryos

    OpenAIRE

    Jaroudi, S.

    2010-01-01

    DNA repair genes are expressed in mammalian embryos and in human germinal vesicles, however, little is known about DNA repair in human preimplantation embryos. This project had three aims: 1) to produce a DNA repair profile of human MII oocytes and blastocysts using expression arrays and identify repair pathways that may be active before and after embryonic genome activation; 2) to design an in vitro functional assay that targeted mismatch repair and which could be applied to human oocytes...

  2. Expression of connexins in human preimplantation embryos in vitro

    Directory of Open Access Journals (Sweden)

    Leese Henry J

    2004-06-01

    Full Text Available Abstract Intercellular communication via gap junctions is required to coordinate developmental processes in the mammalian embryo. We have investigated if the connexin (Cx isoforms known to form gap junctions in rodent preimplantation embryos are also expressed in human embryos, with the aim of identifying species differences in communication patterns in early development. Using a combination of polyA PCR and immunocytochemistry we have assessed the expression of Cx26, Cx31, Cx32, Cx40, Cx43 and Cx45 which are thought to be important in early rodent embryos. The results demonstrate that Cx31 and Cx43 are the main connexin isoforms expressed in human preimplantation embryos and that these isoforms are co-expressed in the blastocyst. Cx45 protein is expressed in the blastocyst but the protein may be translated from a generally low level of transcripts: which could only be detected in the PN to 4-cell embryos. Interestingly, Cx40, which is expressed by the extravillous trophoblast in the early human placenta, was not found to be expressed in the blastocyst trophectoderm from which this tissue develops. All of the connexin isoforms in human preimplantation embryos are also found in rodents pointing to a common regulation of these connexins in development of rodent and human early embryos and perhaps other species.

  3. [Ethical considerations on human cloning. A psychoanalytic perspective].

    Science.gov (United States)

    Alvarez, A

    2000-01-01

    A brief review of ethical issues related to two types of human cloning is presented: cloning embryonic cells not intended to culminate in the birth of a new individual and cloning human beings. Advantages and objections related to both types of human cloning are analyzed from an ethical point of view. Repercussions on individuals born by the technique of cloning are discussed from a psychoanalytical perspective. It can be concluded that cloning embryonic cells could be admissible, while not cloning considered as a reproductive option.

  4. Genetic modification of preimplantation embryos: toward adequate human research policies.

    Science.gov (United States)

    Dresser, Rebecca

    2004-01-01

    Citing advances in transgenic animal research and setbacks in human trials of somatic cell genetic interventions, some scientists and others want to begin planning for research involving the genetic modification of human embryos. Because this form of genetic modification could affect later-born children and their offspring, the protection of human subjects should be a priority in decisions about whether to proceed with such research. Yet because of gaps in existing federal policies, embryo modification proposals might not receive adequate scientific and ethical scrutiny. This article describes current policy shortcomings and recommends policy actions designed to ensure that the investigational genetic modification of embryos meets accepted standards for research on human subjects.

  5. THE CLONING OF HUMAN NEUROTROPHIN-3 GENE

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    In the present study, we have cloned the gene of human neurotrophin-3 (hNT-3) from the genomic DNA of white blood cells (WBC) by polymerase chain reaction (PCR). The amplification products were cloned into pUC19 and sequenced. Genomic sequence comparison of the cloned fragment and the reported hNT-3 (GenBank M61180) reveals 7 base differences: 1 in the signal peptide, 3 in the prepro peptide, and 3 in the mature hNT-3. Except the 2 varied bases (16th, T to G; 285th, A to C) in the signal peptide and pro-sequence resulted in the change of their encoded amino-acids (Tyr→Asp; Gln→His), the other varied bases have no influence on their respective encoded amino-acids, and all the changes have no influence on the open reading frame (ORF) of the hNT-3.

  6. Cloning for human reproduction: one American perspective.

    Science.gov (United States)

    Chester, R

    2001-09-01

    The author, an American law professor, believes that whole-body cloning of adult humans will be possible in the near future. He does not believe the procedure should be banned when used as a form of assisted reproduction, but that it should be regulated by the government to ensure proper testing and application. After raising a number of scientific, ethical, religious and legal issues, Professor Chester addresses parentage in light of both old and new concepts of the 'family.' Finally, he focuses on the problem of women as surrogate mothers of clones, arguing in the process that the surrogate, having no real genetic tie to the clone, would have less of a claim to parentage than at least some of the surrogates currently gestating foetuses.

  7. Cloning humans? Biological, ethical, and social considerations.

    Science.gov (United States)

    Ayala, Francisco J

    2015-07-21

    There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

  8. The impact of preimplantation genetic diagnosis on human embryos

    Directory of Open Access Journals (Sweden)

    García-Ferreyra J.

    2016-12-01

    Full Text Available Chromosome abnormalities are extremely common in human oocytes and embryos and are associated with a variety of negative outcomes for both natural cycles and those using assisted reproduction techniques. Aneuploidies embryos may fail to implant in the uterus, miscarry, or lead to children with serious medical problems (e.g., Down syndrome. Preimplantation genetic diagnosis (PGD is a technique that allows the detection of aneuploidy in embryos and seeks to improve the clinical outcomes od assisted reproduction treatments, by ensuring that the embryos chosen for the transfer are chromosomally normal.

  9. Morphometric analysis of human embryos to predict developmental competence

    DEFF Research Database (Denmark)

    Ziebe, Søren

    2013-01-01

    , but rather choosing and prioritising between the available embryos. Data suggest that only approximately 5% of aspirated human oocytes have the competence to implant and develop into a child and that, in most treatment cycles, there is no oocyte capable of implanting. The most likely outcome is a negative......Morphometric and morphokinetic approaches toward embryo quality assessment have for many years been difficult due to technical limitations. Today, with improvements in laboratory techniques and subsequent quality, we have a better understanding of the morphometric and kinetics of embryo development....... Fertility clinics are moving from "sensing" embryo quality to measuring embryo quality--and this is happening every day in fertility clinics all over the world. However, we cannot select for something that is not there. In daily clinical life it is almost never a question of selecting the optimal embryo...

  10. Microfluidic protocol for in vitro culture of human embryos

    NARCIS (Netherlands)

    Hao, Zhenxia; Kieslinger, D.C.; Vergouw, C.; Kostelijk, H.; Lambalk, C.B.; Le Gac, S.; Zengerle, R.

    2013-01-01

    In vitro culture of pre-implantation embryos is a key-step in Assisted Reproductive Technologies (ART) protocols. In present work we examine the potential of microfluidic devices for pre-implantation human embryo culture, in comparison with conventional droplet-based culture (control). Donated froze

  11. Chromosomal mosaicism in human preimplantation embryos : a systematic review

    NARCIS (Netherlands)

    van Echten-Arends, Jannie; Mastenbroek, Sebastiaan; Sikkema-Raddatz, Birgit; Korevaar, Johanna C.; Heineman, Maas Jan; van der Veen, Fulco; Repping, Sjoerd

    2011-01-01

    BACKGROUND: Although chromosomal mosaicism in human preimplantation embryos has been described for almost two decades, its exact prevalence is still unknown. The prevalence of mosaicism is important in the context of preimplantation genetic screening in which the chromosomal status of an embryo is d

  12. Chromosomal mosaicism in human preimplantation embryos: a systematic review.

    NARCIS (Netherlands)

    Echten-Arends, J. van; Mastenbroek, S.; Sikkema-Raddatz, B.; Korevaar, J.C.; Heineman, M.J.; Veen, F. van der; Repping, S.

    2011-01-01

    BACKGROUND: Although chromosomal mosaicism in human preimplantation embryos has been described for almost two decades, its exact prevalence is still unknown. The prevalence of mosaicism is important in the context of preimplantation genetic screening in which the chromosomal status of an embryo is d

  13. Genetic Modification of Preimplantation Embryos: Toward Adequate Human Research Policies

    OpenAIRE

    Dresser, Rebecca

    2004-01-01

    Citing advances in transgenic animal research and setbacks in human trials of somatic cell genetic interventions, some scientists and others want to begin planning for research involving the genetic modification of human embryos. Because this form of genetic modification could affect later-born children and their offspring, the protection of human subjects should be a priority in decisions about whether to proceed with such research. Yet because of gaps in existing federal policies, embryo mo...

  14. Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Yunhai; PAN; Dengke; SUN; Xiuzhu; SUN; Guojie; WANG; Xiaobo; LIU; Xiaohui; LI; Yan; DAI; Yunping; LI; Ning

    2006-01-01

    In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O2) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O2) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.3%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MⅡ oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.

  15. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos

    Science.gov (United States)

    Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D. Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  16. Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

    Science.gov (United States)

    Qi, Shu-Tao; Liang, Li-Feng; Xian, Ye-Xing; Liu, Jian-Qiao; Wang, Weihua

    2014-01-01

    Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

  17. Ethical questions concerning research on human embryos, embryonic stem cells and chimeras.

    Science.gov (United States)

    Bobbert, Monika

    2006-12-01

    Research using human embryos and embryonic stem cells is viewed as important for various reasons. Apart from questions concerning legal regulations, numerous ethical objections are raised pertaining to the use of surplus embryos from reproductive medicine as well as the creation of embryos and stem cells through cloning. In the hopes of avoiding ethical problems, alternatives have been proposed including the extraction of egg cells from "dead" embryos derived from in vitro fertilization procedures, the extraction of pluripotent stem cells from blastocysts, technologies such as "altered nuclear transfer" (ANT) and "oocyte-assisted reprogramming" (ANT-OAR) as well as parthenogenesis. Initial ethical assessments show that certain questions pertaining to such strategies have remained unanswered. Furthermore, with the help of new or more differentiated biotechnological procedures, it is possible to create chimeras and hybrids in which human and non-human cells are combined. Human-animal chimeras, in which gametes or embryonic tissue have been mixed with embryonic or adult stem cells, demonstrate a different "quality" and "degree of penetration" from those produced in previous experiments. Not only does this have consequences regarding questions of patentability, this situation also raises fundamental questions concerning the human being's self image, the concept of person, identity and species and the moral rights and duties that are connected with such concepts. There is a need for legal regulation, on the national as well as the international level.

  18. Patenting humans: clones, chimeras, and biological artifacts.

    Science.gov (United States)

    Hurlbut, William B

    2005-01-01

    The momentum of advances in biology is evident in the history of patents on life forms. As we proceed forward with greater understanding and technological control of developmental biology there will be many new and challenging dilemmas related to patenting of human parts and partial trajectories of human development. These dilemmas are already evident in the current conflict over the moral status of the early human embryo. In this essay, recent evidence from embryological studies is considered and the unbroken continuity of organismal development initiated at fertilization is asserted as clear and reasonable grounds for moral standing. Within this frame of analysis, it is proposed that through a technique of Altered Nuclear Transfer, non-organismal entities might be created from which embryonic stem cells could be morally procured. Criteria for patenting of such non-organismal entities are considered.

  19. The fate of the mosaic embryo: Chromosomal constitution and development of Day 4, 5 and 8 human embryos

    NARCIS (Netherlands)

    M.A. Santos; G. Teklenburg (Gijs); N.S. Macklon (Nick); D. van Opstal (Diane); G.H. Schuring-Blom (Heleen); P-J. Krijtenburg (Pieter-Jaap); J. de Vreeden-Elbertse (Johanna); B.C.J.M. Fauser (Bart); E.B. Baart (Esther)

    2010-01-01

    textabstractBackground: Post-zygotic chromosome segregation errors are very common in human embryos after in vitro fertilization, resulting in mosaic embryos. However, the significance of mosaicism for the developmental potential of early embryos is unknown. We assessed chromosomal constitution and

  20. Can artificial parthenogenesis sidestep ethical pitfalls in human therapeutic cloning? An historical perspective

    Science.gov (United States)

    Fangerau, H

    2005-01-01

    The aim of regenerative medicine is to reconstruct tissue that has been lost or pathologically altered. Therapeutic cloning seems to offer a method of achieving this aim; however, the ethical debate surrounding human therapeutic cloning is highly controversial. Artificial parthenogenesis—obtaining embryos from unfertilised eggs—seems to offer a way to sidestep these ethical pitfalls. Jacques Loeb (1859–1924), the founding father of artificial parthogenesis, faced negative public opinion when he published his research in 1899. His research, the public's response to his findings, and his ethical foundations serve as an historical argument both for the communication of science and compromise in biological research. PMID:16319240

  1. Developing a code of ethics for human cloning.

    Science.gov (United States)

    Collmann, J; Graber, G

    2000-01-01

    Under what conditions might the cloning of human beings constitute an ethical practice? A tendency exists to analyze human cloning merely as a technical procedure. As with all revolutionary technological developments, however, human cloning potentially exists in a broad social context that will both shape and be shaped by the biological techniques. Although human cloning must be subjected to technical analysis that addresses fundamental ethical questions such as its safety and efficacy, questions exist that focus our attention on broader issues. Asserting that cloning inevitably leads to undesirable consequences commits the fallacy of technological determinism and untenably separates technological and ethical evaluation. Drawing from the Report of the National Bioethics Advisory Committee and Aldous Huxley's Brave New World, we offer a draft "Code of Ethics for Human Cloning" in order to stimulate discussion about the ethics of the broader ramifications of human cloning as well as its particular technological properties.

  2. Ethical issues regarding human cloning: a nursing perspective.

    Science.gov (United States)

    Dinç, Leyla

    2003-05-01

    Advances in cloning technology and successful cloning experiments in animals raised concerns about the possibility of human cloning in recent years. Despite many objections, this is not only a possibility but also a reality. Human cloning is a scientific revolution. However, it also introduces the potential for physical and psychosocial harm to human beings. From this point of view, it raises profound ethical, social and health related concerns. Human cloning would have an impact on the practice of nursing because it could result in the creation of new physiological and psychosocial conditions that would require nursing care. The nursing profession must therefore evaluate the ethics of human cloning, in particular the potential role of nurses. This article reviews the ethical considerations of reproductive human cloning, discusses the main reasons for concern, and reflects a nursing perspective regarding this issue.

  3. The endometrial factor in human embryo implantation

    NARCIS (Netherlands)

    Boomsma, C.M.

    2009-01-01

    The studies presented in this thesis aimed to explore the role of the endometrium in the implantation process. At present, embryo implantation is the major rate-limiting step for success in fertility treatment. Clinicians have sought to develop clinical interventions aimed at enhancing implantation

  4. The endometrial factor in human embryo implantation

    NARCIS (Netherlands)

    Boomsma, C.M.

    2009-01-01

    The studies presented in this thesis aimed to explore the role of the endometrium in the implantation process. At present, embryo implantation is the major rate-limiting step for success in fertility treatment. Clinicians have sought to develop clinical interventions aimed at enhancing implantation

  5. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs.

    Science.gov (United States)

    Kim, M J; Oh, H J; Park, J E; Hong, S G; Kang, J T; Koo, O J; Kang, S K; Jang, G; Lee, B C

    2010-08-01

    To determine factors that affect the efficiency of dog cloning by somatic cell nuclear transfer, the present study was performed to investigate 1) the effects of surgical history (non-operated/operated) and parity (nullipara/multipara) on the recovery of in vivo canine oocytes; 2) the effects of surgical history and parity of recipients on the pregnancy and delivery; and 3) the effects of synchronization state (AA, advanced asynchrony; SY, synchrony; RA, retarded asynchrony) between oocytes donor and recipient on the pregnancy and delivery. Oocyte recovery rate was significantly higher in non-operated dogs compared to operated dogs (93.8 vs. 89.6%, P dogs and multiparous dogs. Delivery rate was also significantly higher in non-operated dogs compared to operated dogs (2.8 vs. 1.0%, P dogs than multiparous dogs (3.0 vs. 1.7%, P dogs as experimental dogs and nulliparous dogs as recipient dogs to increase delivery rate after transfer of somatic cell nuclear transferred embryos, but further study is needed to find out appropriate synchrony status at the transfer.

  7. Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

    OpenAIRE

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-01

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  8. Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

    Science.gov (United States)

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-08

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  9. PiggyBac Transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

    Institute of Scientific and Technical Information of China (English)

    Luo Yi-bo; Zhang Li; Zhu Jiang; Wu Mei-ling; Huan Yan-jun; Yin Zhi; Mu Yan-shuang; Xia Ping; LiuZhong-hua

    2012-01-01

    PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.

  10. In vitro fertilization and stem cell harvesting from human embryos: the law and practice in the United States.

    Science.gov (United States)

    Hook, C Christopher

    2010-07-01

    The challenges before science and medicine are these: science must explore the natural world as thoroughly as possible, while still honoring, protecting, serving and preserving the subject of its investigations, and the human beings for whom it is a tool; medicine must confront disease and disability as effectively as possible, while also honoring, protecting, and preserving those beings for whom it serves - all of those beings, not just some, or even most, at the potential expense of others. These goals are challenged by embryo-destructive human embryonic stem cell research. The human embryo is a human being as clearly defined by embryology, and as such should be protected by the codes governing human subject research. However, because of the "potential" benefits offered by pluripotent stem cells, coupled with abortion politics and a very poorly regulated infertility industry, United States governmental advisory commissions and the scientific, medical, and political communities have attempted to define away the humanity of the human embryo, with a few notable exceptions. Because infertility treatments in the United States are poorly regulated, there are large numbers of supernumerary embryos in cryopreservation. However, only a tiny portion of these will ever be potentially available for research, and thus are not a realistic source of the cells necessary to provide treatments to the millions who might benefit from proposed stem cell based therapies. Cloning will not be the answer either, given the millions of women who must be exploited to provide sufficient numbers of eggs to generate the cloned cell lines. Moreover, the disposition decisions parents must make for their extra embryos are often agonizing, and not uncommonly change. The use of supernumerary embryos as a source for human embryonic stem cells is unethical, will never be a sufficient source for the medical treatments expected from stem cell research, and is often a source of great distress for the

  11. In vitro fertilization and stem cell harvesting from human embryos: the law and practice in the United States

    Directory of Open Access Journals (Sweden)

    C. Christopher Hook

    2010-07-01

    Full Text Available The challenges before science and medicine are these: science must explore the natural world as thoroughly as possible, while still honoring, protecting, serving and preserving the subject of its investigations, and the human beings for whom it is a tool; medicine must confront disease and disability as effectively as possible, while also honoring, protecting, and preserving those beings for whom it serves – all of those beings, not just some, or even most, at the potential expense of others. These goals are challenged by embryo-destructive human embryonic stem cell research. The human embryo is a human being as clearly defined by embryology, and as such should be protected by the codes governing human subject research. However, because of the “potential” benefits offered by pluripotent stem cells, coupled with abortion politics and a very poorly regulated infertility industry, United States governmental advisory commissions and the scientific, medical, and political communities have attempted to define away the humanity of the human embryo, witha few notable exceptions. Because infertility treatments in the United States are poorly regulated, there are large numbersof supernumerary embryos in cryopreservation. However, only a tiny portion of these will ever be potentially available for research, and thus are not a realistic source of the cells necessary to provide treatments to the millions who might benefit from proposed stem cell based therapies. Cloning willnot be the answer either, given the millions of women who must be exploited to provide sufficient numbers of eggs to generate the cloned cell lines. Moreover, the disposition decisions parents must make for their extra embryos are often agonizing, and not uncommonly change.The use of supernumerary embryos as a source for human embryonic stem cells is unethical, will never be a sufficient source for the medical treatments expected from stem cell research, and is often a source of

  12. Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

    Science.gov (United States)

    Akshey, Y S; Malakar, D; De, A Kumar; Jena, M Kumar; Pawar, S Kumar; Dutta, R; Sahu, S

    2011-05-01

    The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with digitonin and Xenopus egg extract

    DEFF Research Database (Denmark)

    Liu, Ying; Østrup, Olga; Li, Juan

    2011-01-01

    Pre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from...... Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells...... cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor...

  14. Thymocyte clones from 14-day mouse embryos. I. State of T cell receptor genes, surface markers, and growth requirements

    OpenAIRE

    1987-01-01

    We have established in culture 13 clones from the thymus of a 14-d B10.BR mouse embryo and characterized 8 of them. All eight FT clones have the TCR-gamma and -beta genes in germline configuration. They express mRNA for the gamma, but not for the beta nor the alpha genes. All eight FT clones are Thy-1+, Ly-1+, LFA-1+, Pgp-1+, H-2K+, and T3-. Three phenotypes could be distinguished on the basis of Lyt-2, L3T4, and IL-2-R expression: Lyt-2+, L3T4-, IL-2-R+ (I); Lyt-2+, L3T4-, IL-2- R- (II); and...

  15. In the world of Dolly, when does a human embryo acquire respect?

    Science.gov (United States)

    Cameron, C; Williamson, R

    2005-04-01

    For most of the 20th century, it was possible to regard fertilisation as the identifiable point when life begins, because this moment could be defined unequivocally and was thought to be the single most essential biological step in the establishment of a new human entity. Since the successful reproductive cloning of Dolly and other mammals, it is clear that any human cell has the potential to supply the full genome of an embryo, and hence a person, without going through fertilisation. At what point in time do such embryos acquire the respect accorded to human beings? The authors argue that the time of implantation is the most useful point at which the potential and the intention to create a new person are translated into reality, because from that point a new life develops. Implantation differentiates a somatic cell in culture (which is not due respect) from a human entity that has acquired its own identity and developmental potential. The authors examine the value of quickening or viability as alternative developmental stages in the process of acquiring respect for the Dolly embryo.

  16. Human cloning: category, dignity, and the role of bioethics.

    Science.gov (United States)

    Shuster, Evelyne

    2003-10-01

    Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?

  17. Keeping up with the cloneses--issues in human cloning.

    Science.gov (United States)

    Rollin, B E

    1999-01-01

    The advent of cloning animals has created a maelstrom of social concern about the "ethical issues" associated with the possibility of cloning humans. When the "ethical concerns" are clearly examined, however, many of them turn out to be less matters of rational ethics than knee-jerk emotion, religious bias, or fear of that which is not understood. Three categories of real and spurious ethical concerns are presented and discussed: 1) that cloning is intrinsically wrong, 2) that cloning must lead to bad consequences, and 3) that cloning harms the organism generated. The need for a rational ethical framework for discussing biotechnological advances is presented and defended.

  18. The Singapore approach to human stem cell research, therapeutic and reproductive cloning.

    Science.gov (United States)

    Kian, Catherine Tay Swee; Leng, Tien Sim

    2005-06-01

    With the controversial ethical issues on the creation of human embryos through cloning for therapeutic research, which holds more promise of medical breakthroughs that the world could ever imagine and the acknowledgement by many scientists that this technology may not lead in the near future to therapies; this country report discusses the approach Singapore takes on human stem cell research, interjected with the authors' own arguments and suggestions especially on research compensation injuries, an often neglected important issue. International comparative viewpoints taken by the major countries in the world are also included in the appendix.

  19. The pros and cons of human therapeutic cloning in the public debate.

    Science.gov (United States)

    Nippert, Irmgard

    2002-09-11

    Few issues linked to genetic research have raised as much controversial debate as the use of somatic cell nuclear transfer technology to create embryos specifically for stem cell research. Whereas European countries unanimously agree that reproductive cloning should be prohibited there is no agreement to be found on whether or not research into therapeutic cloning should be permitted. Since the UK took the lead and voted in favour of regulations allowing therapeutic cloning the public debate has intensified on the Continent. This debate reflects the wide spectrum of diverse religious and secular moralities that are prevalent in modern multicultural European democratic societies. Arguments range from putting forward strictly utilitarian views that weight the moral issues involved against the potential benefits that embryonic stem cell research may harbour to considering the embryo as a human being, endowed with human dignity and human rights from the moment of its creation, concluding that its use for research is unethical and should be strictly prohibited. Given the current state of dissension among the various European states, it is difficult to predict whether 'non-harmonisation' will prevail or whether in the long run 'harmonisation' of legislation that will allow stem cell research will evolve in the EU.

  20. Abnormal Regulation of DNA Methyltransferase Expression in Cloned Mouse Embryos1

    National Research Council Canada - National Science Library

    Young Gie Chung; Sarayu Ratnam; J. Richard Chaillet; Keith E. Latham

    2003-01-01

    Abstract Cloning by somatic cell nuclear transfer is inefficient. This is evident in the significant attrition in the number of surviving cloned offspring at virtually all stages of embryonic and fetal development...

  1. A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos.

    Science.gov (United States)

    Dutta, Rahul; Malakar, Dhruba; Khate, Keviletsu; Sahu, Shailendra; Akshey, Yogesh; Mukesh, Manishi

    2011-09-15

    The main purpose of the experiment was to compare the efficiency of three cell types, namely adult fibroblast, putative embryonic stem (ES) cell, and lymphocyte, as donor cells for somatic cell nuclear transfer by handmade cloning in goats. The outcome clearly shows that putative embryonic stem cells, with a cleavage and blastocyst production rate of 74.69% ± 3.92 and 39.75% ± 3.86, respectively, performs better in comparison to adult fibroblast cell and lymphocyte. Between adult fibroblast cell and lymphocyte no statistically significant difference exists at P II DRB genes of cloned embryos and three donor cells were performed to verify the cloned embryos. The amplified PCR products were subjected to SSCP to confirm their genetic identity. The karyotyping of the cloned embryos showed normal chromosomal status as expected in goat. Significantly, in the second stage of the experiment, the produced cloned embryos were successfully used to derive ntES-like cells. The rate of primary colony formation rate was 62.50% ± 4.62 for fibroblast donor cell derived embryos. The same was 60.60% ± 4.62 for putative ES donor cell derived embryos and 66.66% ± 4.62 for lymphocyte donor cell derived embryos, respectively. The putative ntES colonies were positively characterized for alkaline phosphatase, Oct-4, TRA-1-60, TRA-1-81, Sox-2, and Nanog by Immunocytochemistry and Reverse Transcription PCR. To further validate the stem ness, the produced putative ntES colonies were differentiated to embryoid bodies. Immunocytochemistry revealed that embryoid bodies expressed NESTIN specific for ectodermal lineage; GATA-4 for endodermal lineage and smooth muscle actin-I, and troponin-I specific for mesodermal lineage. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. It could be of substantial significance as patient specific ntES cells have proven therapeutic significance.

  2. Telomeres and the ethics of human cloning.

    Science.gov (United States)

    Allhoff, Fritz

    2004-01-01

    In search of a potential problem with cloning, I investigate the phenomenon of telomere shortening which is caused by cell replication; clones created from somatic cells will have shortened telomeres and therefore reach a state of senescence more rapidly. While genetic intervention might fix this problem at some point in the future, I ask whether, absent technological advances, this biological phenomenon undermines the moral permissibility of cloning.

  3. Human cloning: Eastern Mediterranean Region perspective.

    Science.gov (United States)

    Abdur Rab, M; Khayat, M H

    2006-01-01

    Recent advances in genomics and biotechnology have ushered in a new era in health development. Therapeutic cloning possesses enormous potential for revolutionizing medical and therapeutic techniques. Cloning technology, however, is perceived as having the potential for reproductive cloning, which raises serious ethical and moral concerns. It is important that the Islamic countries come to a consensus on this vital issue. Developing science and technology for better health is a religious and moral obligation. There is an urgent need for Muslim scholars to discuss the issue of stem cell research and cloning rationally; such dialogue will not only consider the scientific merits but also the moral, ethical and legal implications.

  4. Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

    Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

  5. Natural selection of human embryos: decidualizing endometrial stromal cells serve as sensors of embryo quality upon implantation.

    Directory of Open Access Journals (Sweden)

    Gijs Teklenburg

    Full Text Available BACKGROUND: Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1beta, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. CONCLUSIONS: Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits

  6. Activation with ionomycin followed by dehydroleucodine and cytochalasin B for the production of parthenogenetic and cloned bovine embryos.

    Science.gov (United States)

    Canel, Natalia; Bevacqua, Romina; Fernández-Martín, Rafael; Salamone, Daniel F

    2010-08-01

    In this work, Dehydroleucodine (DhL) was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer (SCNT) reconstituted embryos. Oocytes were activated with 5 microM Ionomycin (Io) and exposed for 3 h to 1 or 5 microM DhL alone (Io-Dhl1 or Io-DhL5) or combined with Cytochalasin B (Io-DhL1/CB; Io-DhL5/CB). Control groups were Io (Io), Io followed by 1.9 mM 6-Dimethylaminopurine (Io-6DMAP), and embryos produced by in vitro fertilization (IVF). Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates (1 microM) were karyotyped. An additional treatment based in Io-DhL1 plus lengthened (6-h) exposure to CB (Io-DhL1/long CB) was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 microM DhL than with 5 microM DhL, either for groups combined or not with CB (19.15; 21.74 vs. 6.82; 0%, respectively) (p DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP (35.85%) and IVF (33.33%) groups (p > 0.05). Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP (63.8 vs. 36.8; 40 and 31.6%, respectively) (p DhL treatments resulted in polyploidy rates that were lower than Io-DMAP (5.2, 12.0, 10.6, and 31.6%, respectively) (p DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP (6.1, 9.4, and 18.3%, respectively) (p DhL1/long CB protocol could be useful for SCNT programs.

  7. Reasoning about embryos, cloning and stem cells: let's get more clear and distinct.

    Science.gov (United States)

    Parker, Malcolm

    2003-01-01

    Plural democratic societies encourage and require the tolerance of disparate views. However, in relation to contentious areas like assisted reproductive technologies and destructive embryo research, tolerance is strained by the normative force of our fundamental beliefs about the moral status of early human forms. Yet in the continuing debates, spokespersons for different positions often do not concede all the implications of their arguments, may sidestep the real moral issues, and can fail to be clear about the foundations on which their arguments and policy advice ultimately rely. Guidelines and statutes can be rendered incoherent by the desire to balance and satisfy opposing values, rather than honestly reflecting the primary values they espouse. I call for greater clarity and honesty as these issues continue to be debated. An uncritical adherence to pluralism will encourage strategic obfuscation, but citizens of democracies need to be clearly informed about all the premises of opposing positions. Decisions about ethically and legally acceptable reproductive technologies ultimately depend on support for one metaphysical grounding at the expense of another. This should be acknowledged, as should its implications for policy.

  8. Human reproductive cloning: an analysis of the Andrews Report.

    Science.gov (United States)

    Little, Kim

    2002-01-01

    There is nothing like an overwhelming consensus of opinion to encourage a less than rigourous approach to analyzing complex ethical issues. Unfortunately, this is nowhere more apparent than in the discussion of human reproductive cloning contained in the federal House of Representatives Standing Committee on Legal and Constitutional Affairs' report on human cloning, released last August. The report may well fulfil the first half of its project, namely the empirical task of adequately summarizing and categorizing the various submissions made to the Committee. However, it is clearly inadequate as a discussion of the ethical and legal permissibility of human reproductive cloning.

  9. Successful cloning of the Yucatan minipig using commercial/occidental breeds as oocyte donors and embryo recipients.

    Science.gov (United States)

    Estrada, Jose L; Collins, Bruce; York, Abby; Bischoff, Steve; Sommer, Jeff; Tsai, Shengdar; Petters, Robert M; Piedrahita, Jorge A

    2008-06-01

    The widespread application of porcine SCNT to biomedical research is being hampered by the large adult size (300-600 lbs) of the commercial breeds commonly used for SCNT. The Yucatan minipig, in contrast, has an adult weight of 140-150 lbs and a long history of utility in biomedical research. In order to combine the wide availability of commercial swine with the biomedical value of the Yucatan minipig, we utilized SCNT using the Yucatan as nuclear donors and commercial swine as both oocyte donors and recipients. Of six recipient gilts receiving 631 SCNT embryos, three went to term and delivered seven piglets, four of which survived to adulthood. Additionally, we obtained fetal fibroblasts from a cloned Yucatan and used them for a second round of SCNT. Of three recipients receiving 315 reconstructed embryos, one went to term and delivered three piglets, one of which survived to adulthood. Both microsatellite and D-loop sequence analysis confirmed that all of the piglets generated were nuclear-mitochondrial hybrids carrying Yucatan nuclear DNA and commercial breed mitochondrial DNA. This report shows that it is possible to produce viable Yucatan SCNT clones and opens up the possibility of developing valuable biomedical models in this porcine breed.

  10. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media

    NARCIS (Netherlands)

    Kleijkers, S.H.M.; Eijssen, L.M.T.; Coonen, E.; Derhaag, J.G.; Mantikou, E.; Jonker, M.J.; Mastenbroek, S.; Repping, S.; Evers, J.L.H.; Dumoulin, J.C.M.; van Montfoort, A.P.A.

    2015-01-01

    STUDY QUESTION: Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? SUMMARY ANSWER: Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes inv

  11. Cloning of non-human primates: the road "less traveled by".

    Science.gov (United States)

    Sparman, Michelle L; Tachibana, Masahito; Mitalipov, Shoukhrat M

    2010-01-01

    Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.

  12. Cloning of non-human primates: the road “less traveled by”

    Science.gov (United States)

    SPARMAN, MICHELLE L.; TACHIBANA, MASAHITO; MITALIPOV, SHOUKHRAT M.

    2011-01-01

    Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model. PMID:21404187

  13. The global governance of human cloning: the case of UNESCO

    Science.gov (United States)

    Langlois, Adèle

    2017-01-01

    Since Dolly the Sheep was cloned in 1996, the question of whether human reproductive cloning should be banned or pursued has been the subject of international debate. Feelings run strong on both sides. In 2005, the United Nations adopted its Declaration on Human Cloning to try to deal with the issue. The declaration is ambiguously worded, prohibiting “all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life”. It received only ambivalent support from UN member states. Given this unsatisfactory outcome, in 2008 UNESCO (the United Nations Educational, Scientific and Cultural Organization) set up a Working Group to investigate the possibility of a legally binding convention to ban human reproductive cloning. The Working Group was made up of members of the International Bioethics Committee, established in 1993 as part of UNESCO’s Bioethics Programme. It found that the lack of clarity in international law is unhelpful for those states yet to formulate national regulations or policies on human cloning. Despite this, member states of UNESCO resisted the idea of a convention for several years. This changed in 2015, but there has been no practical progress on the issue. Drawing on official records and first-hand observations at bioethics meetings, this article examines the human cloning debate at UNESCO from 2008 onwards, thus building on and advancing current scholarship by applying recent ideas on global governance to an empirical case. It concludes that, although human reproductive cloning is a challenging subject, establishing a robust global governance framework in this area may be possible via an alternative deliberative format, based on knowledge sharing and feasibility testing rather than the interest-based bargaining that is common to intergovernmental organizations and involving a wide range of stakeholders. This article is published as part of a collection on global governance.

  14. The global governance of human cloning: the case of UNESCO.

    Science.gov (United States)

    Langlois, Adèle

    2017-03-21

    Since Dolly the Sheep was cloned in 1996, the question of whether human reproductive cloning should be banned or pursued has been the subject of international debate. Feelings run strong on both sides. In 2005, the United Nations adopted its Declaration on Human Cloning to try to deal with the issue. The declaration is ambiguously worded, prohibiting "all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life". It received only ambivalent support from UN member states. Given this unsatisfactory outcome, in 2008 UNESCO (the United Nations Educational, Scientific and Cultural Organization) set up a Working Group to investigate the possibility of a legally binding convention to ban human reproductive cloning. The Working Group was made up of members of the International Bioethics Committee, established in 1993 as part of UNESCO's Bioethics Programme. It found that the lack of clarity in international law is unhelpful for those states yet to formulate national regulations or policies on human cloning. Despite this, member states of UNESCO resisted the idea of a convention for several years. This changed in 2015, but there has been no practical progress on the issue. Drawing on official records and first-hand observations at bioethics meetings, this article examines the human cloning debate at UNESCO from 2008 onwards, thus building on and advancing current scholarship by applying recent ideas on global governance to an empirical case. It concludes that, although human reproductive cloning is a challenging subject, establishing a robust global governance framework in this area may be possible via an alternative deliberative format, based on knowledge sharing and feasibility testing rather than the interest-based bargaining that is common to intergovernmental organizations and involving a wide range of stakeholders. This article is published as part of a collection on global governance.

  15. A review of Greek law on human cloning.

    Science.gov (United States)

    Mavroforou, Anna; Giannoukas, Athanasios; Michalodimitrakis, Emmanuel

    2003-01-01

    The creation of Dolly, a cloned lamb from adult cells was a major scientific breakthrough, which opened new avenues for many research fields such as reproductive medicine, transplantation and biotechnology. However this achievement brought to public attention the theoretical possibility of human reproductive cloning. Inevitably heated debate occurred on several ethical and legal consequences of the prospect of human cloning. At the present time there is no legal framework in any country to respond to this challenge in a pragmatic way in order to protect human rights and at the same time to allow science to work for the best interests of mankind. Greece is a European Union country with its own traditions, history, culture and beliefs but without political and legislative experience in the handling of medical and biotechnological matters. This paper aims to discuss the legal issues likely to be raised by the prospect of human reproductive cloning in relation to the current state of the Greek legal system.

  16. Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

    Science.gov (United States)

    Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

    2012-01-01

    Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

  17. Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

    Institute of Scientific and Technical Information of China (English)

    YAN Jie; ZHU Guijin; LIU Jianxin; AI Jihui

    2000-01-01

    An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0.4%bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73% developed to the morula stage and 67.21% cavitated to blastocysts with 59.74 % hatching, as compared with 61.34% to morula stage, 48.47% to blastocysts and none hatching in the controls,respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

  18. Evidence for a human-specific Escherichia coli clone.

    Science.gov (United States)

    Clermont, Olivier; Lescat, Mathilde; O'Brien, Claire L; Gordon, David M; Tenaillon, Olivier; Denamur, Erick

    2008-04-01

    Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.

  19. Analysis of an epigenetic argument against human reproductive cloning.

    Science.gov (United States)

    Nordgren, Anders

    2006-08-01

    Human reproductive cloning is a much disputed ethical issue. This technology is often condemned as being contrary to human dignity. However, there are also risk arguments. An ethical argument that is often put forward by scientists but seldom developed in more detail focuses on health risks in animal cloning. There is a high risk that animal clones exhibit abnormalities and these are increasingly believed to be due to errors in epigenetic reprogramming. The argument is that human reproductive cloning should not be carried out because human clones are also likely to exhibit abnormalities due to inappropriate epigenetic reprogramming. Different versions of this epigenetic argument are analysed, a categorical version and a non-categorical. The non-categorical version is suggested to be more well-considered. With regard to policy making on human reproductive cloning, the categorical version can be used to prescribe a permanent ban, while the non-categorical version can be used to prescribe a temporary ban. The implications of the precautionary principle--as interpreted in the European Union--are investigated. The conclusion is that it seems possible to support a temporary ban by reference to this principle.

  20. In vitro produced and cloned embryos: Effects on pregnancy, parturition and offspring

    NARCIS (Netherlands)

    Kruip, T.A.M.; den Daas, J.H.G.

    1997-01-01

    Earlier reports have indicated that the transfers of bovine and ovine embryos produced by in vitro procedures (IVP) or by nuclear transfer (NT) have resulted in the birth of heavy offspring. The present paper presents summary information from 30 data sets obtained worldwide (WW) on IVP and NT in dif

  1. Cloning and characterization of the neural isoforms of human dystonin

    Energy Technology Data Exchange (ETDEWEB)

    Brown, A.; Dalpe, G.; Mathieu, M.; Kothary, R. [Univ. of Montreal, Quebec (Canada)

    1995-10-10

    Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5{prime} exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical. 17 refs., 2 figs.

  2. Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex.

    Science.gov (United States)

    Makino, Hatsune; Yamazaki, Yukiko; Hirabayashi, Takahiro; Kaneko, Ryosuke; Hamada, Shun; Kawamura, Yoshimi; Osada, Tomoharu; Yanagimachi, Ryuzo; Yagi, Takeshi

    2005-01-01

    Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.

  3. Clonal propagation of primate offspring by embryo splitting.

    Science.gov (United States)

    Chan, A W; Dominko, T; Luetjens, C M; Neuber, E; Martinovich, C; Hewitson, L; Simerly, C R; Schatten, G P

    2000-01-14

    Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.

  4. In Vitro Developmental Potential of Cloned Embryos Derived from Bovine Somatic Cells and Rabbits Oocyte

    Institute of Scientific and Technical Information of China (English)

    LIU Ya; LI Bin; ZHAO Huan; CHENG Li-zi; ZHANG Xiao-rong; CHEN Da-yuan; ZHANG Yun-hai; ZHANG Zhi-guo; JING Ren-tao; WANG Cun-li; ZHANG Mei-lin; LI Dong-wei

    2003-01-01

    180 reconstituted embryos were produced by nuclear transplantation using bovine ear fibroblasts at G0 or non-G0 stage as donor nuclei and oocytes collected from superovulated multiparous or young rabbits as recipients. After cultivation in two kinds of medium M199+ 10%FBS or RD+ 10%FBS, 112 of them developed to 2-cell stage (62.2%) and 26 to morula stage (14.4%) and 20 of them eventually developed to blastocyst stage (11. 1% ). There is no significant difference for the cleavage rates in two groups of reconstituted embryos derived from G0-stage and non-G0 stage donor cells respectively. However, G0-stage donor cells could result in higher rate of 8-cell - 16-cell stage embryos significantly (P<0.05), as well as higher rate of blastocysts (P<0.01). It seems that using two different culture systems had no significant effects on the cleavage rate, morula rate or blastocyst rate (P>0.05).

  5. Pathogenesis, developmental consequences, and clinical correlations of human embryo fragmentation.

    Science.gov (United States)

    Fujimoto, Victor Y; Browne, Richard W; Bloom, Michael S; Sakkas, Denny; Alikani, Mina

    2011-03-15

    This narrative review summarizes the current state of knowledge about human embryo fragmentation during IVF. The clinical relevance of fragmentation is discussed and evidence supporting a central role for the oocyte in the pathogenesis of fragmentation is presented. A mechanism of fragmentation as aberrant cell division involving the cytoskeleton is described along with the novel concept of membrane instability in relation to follicular high-density lipoprotein metabolism and cholesterol transport. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  7. Origin of the hematopoietic system in the human embryo.

    Science.gov (United States)

    Julien, Emmanuelle; El Omar, Reine; Tavian, Manuela

    2016-11-01

    The continuous generation of blood cells throughout life relies on the existence of hematopoietic stem cells (HSC) generated during embryogenesis. Given the importance of HSC transplantation in cell-based therapeutic approaches, considerable efforts have been made toward understanding the developmental origins of embryonic HSC. Adult-type HSC are first generated in the aorta-gonad-mesonephros (AGM) region between days 27 and 40 of human embryonic development, but an elusive blood-forming potential is present earlier in the underlying splanchnopleura. It is relatively well accepted that the HSC emerge in the AGM through a hemogenic endothelium, but the direct precursor of this cell type remains to be clearly identified. This review is intended to summarize the recent advances made to understand the origins of hematopoietic stem cells in the early human embryo. In addition, we discuss in detail the discovery of the angiotensin-converting enzyme (ACE) as a novel marker of human HSC and of prehematopoietic precursors inside the embryo. © 2016 Federation of European Biochemical Societies.

  8. Effects of high hydrostatic pressure on genomic expression profiling of porcine parthenogenetic activated and cloned embryos

    DEFF Research Database (Denmark)

    Lin, Lin; Luo, Yonglun; Sørensen, Peter

    2014-01-01

    Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular...

  9. [The status of human cloning in the international setting].

    Science.gov (United States)

    Rey del Castillo, Javier

    2006-01-01

    The General Assembly of the United Nations submitted a Declaration on Human Cloning in March 2005. The text of such Declaration was the result of a difficult and long process, taking more than three years. Being a Declaration instead of a Resolution, it has not legal capability in inforcing United Nations members to act according to its recommendations. This article begins with an explanation of several terms referred to cloning. Different countries' legislation on cloning is analyzed. Positions of the same countries at the Convention of the United Nations are as well analyzed. Comparing both countries' views shows that national legislation on cloning is independent and orientated by some countries' particular interests and biological and ethical views on these issues. Future developments on human cloning and its applications will be shared among all countries, both the ones currently allowing and supporting "therapeutic" cloning and the ones now banning it. In such case, it would be important to reach agreements on these issues at an international level. The article discusses possible legislative developments and offers some proposals to reach such agreements.

  10. The moral status of the embryo: the human embryo in the UK Human Fertilisation and Embryology (Research Purposes) Regulation 2001 debate.

    Science.gov (United States)

    Bahadur, G

    2003-01-01

    The use of the embryo in research into birth defects, infertility and the possible therapeutic value of embryonic stem cells, has given rise to vigorous discussion of the ethical, moral and legal status of the embryo. This paper considers the parliamentary debate that surrounded the passing of legislation in the UK in 2000 governing the use of the embryo in research. Underlying disagreement by members of Parliament as to whether embryo research was permissible, were considerable differences regarding when life was thought to begin--whether at the moment of fertilization of the egg, or whether after 14 days, at the time of the beginnings of cell differentiation, and the point after which the embryo can no longer split to form twins. Those who favoured the latter view argued that, while the conceptus might possess a unique genetic formula, it had only the potential for life before 14 days, the development of human life being a gradual and continuous process. They considered it mistaken to accord the embryo full human rights. Those who adopted an opposed standpoint insisted that life was present and actual from the moment of conception and therefore sacrosanct and inviolable. The notion of the pre-embryo, they maintained, merely serves to disguise the embryo's humanity.

  11. United Nations and human cloning: a slender and fortunate defence for biomedical research.

    Science.gov (United States)

    Edwards, R G

    2003-12-01

    Numerous biomedical scientists have contributed to the wide knowledge on the growth of preimplantation human embryos in vitro, now improving every aspect of the form of clinical care. These data were gained ethically in many countries, to open new vistas including the alleviation of infertility, preimplantation genetic diagnosis and stem cells, combined with some recent reports on human reproductive cloning. After detailed consultations with scientists, clinicians, ethicists and lawyers, many governments passed legislation permitting research under their own particular socially-defined conditions. Virtually all of them rejected reproductive cloning; a few have accepted therapeutic cloning. These legislatures saluted the many biomedical scientists striving to improve IVF and its derivatives, recognizing their immense medical potential. A motion recently placed before the United Nations then recommended a worldwide ban on all forms of human cloning. Proponents included the Vatican and many Roman Catholic countries, the USA and others. Opponents included Belgium, China, Japan, Brazil, UK, Germany and France. Mediation was achieved by Iran and other Muslim nations, and led to a motion passed by single vote for a two-year delay. This may be the first-ever proposal to ban worldwide a particular form of research. It sounds the alarm bells for further research. It raises questions about the UN being an appropriate forum for ethical decisions affecting the entire world and its future medicine. Large blocs of nations committed to particular religions and outlooks confronted each other, a situation in total contrast to the detailed and widespread consultations made by individual governments when deciding their own individual ethics. This event was clearly a narrow escape for free research as defined by each country's own jurisprudence. It also places research on human embryology and reproductive biomedicine into a more critical situation than before. Current liberalism in

  12. Trichostatin A modified histone covalent pattern and enhanced expression of pluripotent genes in interspecies black-footed cat cloned embryos but did not improve in vitro and in vivo viability.

    Science.gov (United States)

    Gómez, Martha C; Pope, C Earle; Biancardi, Monica N; Dumas, Cherie; Galiguis, Jason; Morris, Anna Claire; Wang, Guoshun; Dresser, Betsy L

    2011-08-01

    Abstract The black-footed cat (BFC; Felis nigripes), one of the smallest wild cats, is listed as threatened. Interspecies somatic cell nuclear transfer (Is-SCNT) offers the possibility of preserving endangered species. Development to term of interspecies BFC (Is-BFC) cloned embryos has not been obtained, possibly due to abnormal epigenetic reprogramming. Treatment of intraspecies cloned embryos with TSA improves nuclear reprogramming and in vitro and in vivo viability. In this study, we evaluated (1) whether covalent histone modifications differ between Is-BFC cloned embryos and their IVF counterparts, (2) the optimal TSA concentration and exposure times to modify the covalent histone patterns, (3) if TSA enhances in vitro and in vivo developmental competence of cloned embryos, and (4) expression of pluripotent genes. Results indicated that the covalent histone modifications of Is-BFC cloned embryos aberrantly differ from their DSH-IVF counterpart embryos. Aberrant epigenetic events may be due partially to the inability of the DSH cytoplasm to modify the restrictive epigenetic marks of the BFC nuclei after somatic cell nuclear transfer (SCNT). Incomplete remodeling of the histone H3K9me2 in Is-BFC cloned embryos possibly contributes to abnormal expression of pluripotent genes and low embryonic development. Treatment of Is-BFC cloned embryos with TSA remodeled the covalent pattern in H3K9ac and H3K9me2, resembling epigenetic patterns in IVF counterpart embryos, and resulted in activation of some pluripotent genes. However, genomic reprogramming of Is-BFC cloned blastocysts did not follow the same reprogramming pattern observed in DSH-IVF embryos, and in vitro and in vivo developmental competence was not enhanced.

  13. The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos.

    Science.gov (United States)

    Lee, Eugine; Kim, Ji Hye; Park, Seon Mi; Jeong, Yeon Ik; Lee, Jong Yun; Park, Sun Woo; Choi, Jiho; Kim, Huen Suk; Jeong, Yeon Woo; Kim, Sue; Hyun, Sang Hwan; Hwang, Woo Suk

    2008-05-01

    The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; Pcell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.

  14. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates.

    Science.gov (United States)

    Wong, Kai Mee; Mastenbroek, Sebastiaan; Repping, Sjoerd

    2014-07-01

    Cryopreservation of human embryos is now a routine procedure in assisted reproductive technologies laboratories. There is no consensus on the superiority of any protocol, and substantial differences exist among centers in day of embryo cryopreservation, freezing method, selection criteria for which embryos to freeze, method of embryo thawing, and endometrial preparation for transfer of frozen-thawed embryos. In the past decade, the number of frozen-thawed embryo transfer cycles per started in vitro fertilization (IVF) cycle increased steadily, and at the same time the percentage of frozen-thawed embryo transfers that resulted in live births increased. Currently, cryopreservation of human embryos is more important than ever for the cumulative pregnancy rate after IVF. Interestingly, success rates after frozen-thawed embryo transfer are now nearing the success rates of fresh embryo transfer. This supports the hypothesis of so called freeze-all strategies in IVF, in which all embryos are frozen and no fresh transfer is conducted, to optimize success rates. High-quality randomized controlled trials should be pursued to find out which cryopreservation protocol is best and whether the time has come to completely abandon fresh transfers.

  15. Abnormalities in centrosome number in human embryos and embryonic stem cells.

    Science.gov (United States)

    Gu, Yi-Fan; OuYang, Qi; Dai, Can; Lu, Chang-Fu; Lin, Ge; Gong, Fei; Lu, Guang-Xiu

    2016-05-01

    Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc.

  16. Development of the ventral body wall in the human embryo.

    Science.gov (United States)

    Mekonen, Hayelom K; Hikspoors, Jill P J M; Mommen, Greet; Köhler, S Eleonore; Lamers, Wouter H

    2015-11-01

    Migratory failure of somitic cells is the commonest explanation for ventral body wall defects. However, the embryo increases ~ 25-fold in volume in the period that the ventral body wall forms, so that differential growth may, instead, account for the observed changes in topography. Human embryos between 4 and 10 weeks of development were studied, using amira reconstruction and cinema 4D remodeling software for visualization. Initially, vertebrae and ribs had formed medially, and primordia of sternum and hypaxial flank muscle primordium laterally in the body wall at Carnegie Stage (CS)15 (5.5 weeks). The next week, ribs and muscle primordium expanded in ventrolateral direction only. At CS18 (6.5 weeks), separate intercostal and abdominal wall muscles differentiated, and ribs, sterna, and muscles began to expand ventromedially and caudally, with the bilateral sternal bars fusing in the midline after CS20 (7 weeks) and the rectus muscles reaching the umbilicus at CS23 (8 weeks). The near-constant absolute distance between both rectus muscles and approximately fivefold decline of this distance relative to body circumference between 6 and 10 weeks identified dorsoventral growth in the dorsal body wall as determinant of the 'closure' of the ventral body wall. Concomitant with the straightening of the embryonic body axis after the 6th week, the abdominal muscles expanded ventrally and caudally to form the infraumbilical body wall. Our data, therefore, show that the ventral body wall is formed by differential dorsoventral growth in the dorsal part of the body.

  17. Goat uterine DBA+ leukocytes differentiation and cytokines expression respond differently to cloned versus fertilized embryos.

    Directory of Open Access Journals (Sweden)

    Lijuan Qin

    Full Text Available High rate of fetal mortality in ruminant somatic cell nuclear transfer (SCNT pregnancies is due, at least in part, to immune-mediated abortion of fetuses. In the present study, goat uterine leukocytes were isolated by Dolichos biflorus agglutinin (DBA coated magnetic beads, and with majority being were CD56+CD16- in phenotype with low levels of perforin and Granzyme B expression. The responses of the isolated cells to SCNT and in vitro fertilization (IVF embryos conditioned mediums containing hormone steroids were compared by measuring their phenotype and cytokines expression. The results showed there was a 2-fold increase in the numbers of isolated uterine leukocytes after incubation with different conditioned mediums for 120 h. However, significantly lower percentage and absolute numbers of uterine CD56+CD16- leukocytes incubated with SCNT conditioned mediums were detected as compared with those incubated with IVF conditioned mediums (P < 0.05. The group treated with progesterone (P4 or the combination of P4 and 17β-estradiol (E2 were associated with significantly higher percentage and absolute numbers of CD56+CD16- cells as compared with those treated with E2 alone (P < 0.05. Furthermore, in the presence of steroids, the isolated leukocytes incubated with SCNT conditioned mediums associated with greater levels of IFN-γ secretion and expression, as well as lesser levels of VEGF, as compared with those treated with IVF conditioned mediums (P < 0.05. In conclusion, this study demonstrates that SCNT embryos have a profound effect on the phenotype expression of goat uterine DBA+ leukocytes, as well as the secretion and expression of IFN-γ and VEGF by these cells in vitro.

  18. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    Science.gov (United States)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  19. Psychological aspects of human cloning and genetic manipulation: the identity and uniqueness of human beings.

    Science.gov (United States)

    Morales, N M

    2009-01-01

    Human cloning has become one of the most controversial debates about reproduction in Western civilization. Human cloning represents asexual reproduction, but the critics of human cloning argue that the result of cloning is not a new individual who is genetically unique. There is also awareness in the scientific community, including the medical community, that human cloning and the creation of clones are inevitable. Psychology and other social sciences, together with the natural sciences, will need to find ways to help the healthcare system, to be prepared to face the new challenges introduced by the techniques of human cloning. One of those challenges is to help the healthcare system to find specific standards of behaviour that could be used to help potential parents to interact properly with cloned babies or children created through genetic manipulation. In this paper, the concepts of personality, identity and uniqueness are discussed in relationship to the contribution of twin studies in these areas. The author argues that an individual created by human cloning techniques or any other type of genetic manipulation will not show the donor's characteristics to the extent of compromising uniqueness. Therefore, claims to such an effect are needlessly alarmist.

  20. A role for Aurora C in the chromosomal passenger complex during human preimplantation embryo development

    NARCIS (Netherlands)

    Santos, Margarida Avo; van de Werken, Christine; de Vries, Marieke; Jahr, Holger; Vromans, Martijn J. M.; Laven, Joop S. E.; Fauser, Bart C.; Kops, Geert J.; Lens, Susanne M.; Baart, Esther B.

    2011-01-01

    BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important m

  1. Parliamentary cultures and human embryos: the Dutch and British debates compared

    NARCIS (Netherlands)

    Kirejczyk, Marta

    1999-01-01

    Twenty years ago, the technology of in vitro fertilization created a new artefact: the human embryo outside the woman's body. In many countries, political debates developed around this artefact. One of the central questions in these debates is whether it is permissible to use human embryos in resear

  2. Addressing the ethical issues raised by synthetic human entities with embryo-like features

    NARCIS (Netherlands)

    Aach, John; Lunshof, Jeantine; Iyer, Eswar; Church, George M.

    2017-01-01

    The "14-day rule" for embryo research stipulates that experiments with intact human embryos must not allow them to develop beyond 14 days or the appearance of the primitive streak. However, recent experiments showing that suitably cultured human pluripotent stem cells can self organize and recapitul

  3. Scientific and medical aspects of human reproductive cloning

    National Research Council Canada - National Science Library

    Committee on Science

    2002-01-01

    ...), and the Institute of Medicine (IOM). It is a result of work done by the Panel on Scientific and Medical Aspects of Human Cloning, a joint panel of the Committee on Science, Engineering, and Public Policy (COSEPUP) and the Board on Life Sciences (BLS). The members of the panel responsible for the report were chosen for their special competences and with regard ...

  4. Molecular cloning of the human excision repair gene ERCC-6.

    NARCIS (Netherlands)

    C. Troelstra (Christine); H. Odijk (Hanny); J. de Wit (Jan); A. Westerveld (Andries); L.H. Thompson; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1990-01-01

    textabstractThe UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5

  5. [Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

    Science.gov (United States)

    An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

    2012-12-01

    We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

  6. [Cloning and law in Hungary].

    Science.gov (United States)

    Julesz, Máté

    2015-03-01

    Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

  7. Endocardial tip cells in the human embryo - facts and hypotheses.

    Directory of Open Access Journals (Sweden)

    Mugurel C Rusu

    Full Text Available Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43-56 days were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin, CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.

  8. Effects of donor cells on in vitro development of cloned bovine embryos

    Institute of Scientific and Technical Information of China (English)

    Jing Fu; Pengfei Guan; Leiwen Zhao; Hua Li; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2008-01-01

    The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

  9. Cloning and expression of special F protein from human liver

    Institute of Scientific and Technical Information of China (English)

    Shu-Ye Liu; Xin-Da Yu; Chun-Juan Song; Wei Lu; Jian-Dong Zhang; Xin-Rong Shi; Ying Duan; Ju Zhang

    2007-01-01

    AIM:To clone human liver special F protein and to express it in a prokaryotic system.METHODS:Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this,cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E coli BL21 (DEB) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein.RESULTS:The cDNA clone of human liver special F protein (1134bp) was successfully produced,with the cDNA sequence being published in Gene-bank:DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted.CONCLUSION:F protein expresses cDNA clone in a proKaryotic system,which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.

  10. Analysis of compaction initiation in human embryos by using time-lapse cinematography.

    Science.gov (United States)

    Iwata, Kyoko; Yumoto, Keitaro; Sugishima, Minako; Mizoguchi, Chizuru; Kai, Yoshiteru; Iba, Yumiko; Mio, Yasuyuki

    2014-04-01

    To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

  11. Donating embryos for human embryonic stem cell (hESC) research: a committee opinion.

    Science.gov (United States)

    2013-10-01

    hESC research is an ethically acceptable use of human embryos that are in excess of those needed to meet the fertility goals of patients. The ethical basis for this view and issues to be considered during the informed consent process for the donation of embryos are developed in this document. This report replaces the Committee's 2009 report, "Donating spare embryos for stem cell research" (Fertil Steril 2009;91:667-70).

  12. Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

    Science.gov (United States)

    Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham

    2012-06-01

    This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (pcloning, chimera formation, and transgenic animal production.

  13. Assessment of developmental potential of caprine cloned embryos with ooplasm replenishment under two culture media.

    Science.gov (United States)

    Gopalakrishna, R; Kumar, Dharmendra; Singh, Ajay Pratap; Pandey, Saurabh Kumar; Ranjan, Rakesh; Sarkhel, B C

    2014-03-12

    The present study was designed to assess the developmental potential of somatic cell nuclear transfer (SCNT) embryos, with and without replenishment of ooplasm into enucleated oocytes, by culturing separately in two culture media. The enucleated oocytes were replenished with exogenous matured ooplasm under replenished nuclear transfer (RNT) method and compared with conventional nuclear transfer (CNT) method without replenishment. The fusion efficiency in RNT group was found to be significantly higher (P < 0.05) than CNT group (59.39 ± 7.36 vs 45.57 ± 3.68%). The completely fused reconstructed oocytes from both groups were cultured separately in research vitro cleave medium (RVCL) and RVCL-Blast medium. The embryonic development of 2 cell, 4 cell, 8-16 cell and 16-32 cell stages in the RNT group was superior to the CNT group regardless of the culture media used (P < 0.05). The embryonic development of the 8-16 cell, 16-32 cell, morula, and blastocyst stages in the RVCL-Blast medium was significantly higher (P < 0.05) than the RVCL alone for both RNT as well as CNT groups. RNT method with RVCL-Blast produced highly significant (P < 0.01) embryonic development for 8-16 cell and 16-32 cell stage when compared to CNT with RVCL. Conclusively, the combination of RNT with RVCL-Blast culture media enabled an overall increase in the embryonic developmental potential.

  14. Human Cloning as the Other in Ishiguro's Never Let Me Go

    National Research Council Canada - National Science Library

    Guo, Wen

    2015-01-01

    In her article "Human Cloning as the Other in Ishiguro's Never Let Me Go" Wen Guo analyzes Kazuo Ishiguro's novel with focus on Ishiguro's analogy between human cloning and people of marginality in contemporary society...

  15. New techniques on embryo manipulation.

    Science.gov (United States)

    Escribá, M J; Valbuena, D; Remohí, J; Pellicer, A; Simón, C

    2002-01-01

    For many years, experience has been accumulated on embryo and gamete manipulation in livestock animals. The present work is a review of these techniques and their possible application in human embryology in specific cases. It is possible to manipulate gametes at different levels, producing paternal or maternal haploid embryos (hemicloning), using different techniques including nuclear transfer. At the embryonic stage, considering practical, ethical and legal issues, techniques will be reviewed that include cloning and embryo splitting at the cleavage stage, morula, or blastocyst stage.

  16. Cloning of Integral Mature Peptide Gene of Human GDF-5

    Institute of Scientific and Technical Information of China (English)

    王万山; 顾为望; 王启伟; 朴仲贤; 朴英杰

    2004-01-01

    Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.

  17. Roscovitine treatment improves synchronization of donor cell cycle in G0/G1 stage and in vitro development of handmade cloned buffalo (Bubalus bubalis) embryos.

    Science.gov (United States)

    Selokar, Naresh L; Saini, Monika; Muzaffer, Mushariffa; Krishnakanth, G; Saha, Ambika P; Chauhan, Manmohan S; Manik, Radheysham; Palta, Prabhat; Madan, Pavneesh; Singla, Suresh K

    2012-04-01

    This study investigated the effects of serum-starvation, total confluence, and roscovitine treatment on cell-cycle synchronization of buffalo ear skin fibroblasts to the G0/G1 stage and on the developmental competence of cloned embryos. Serum starvation of total confluence cultures for 24 h had a higher (proscovitine treatment than that with 10 μM (94.4, 96.4, and 86.6%, respectively), which was similar to that for total confluence (86.0%). MTT assay showed that cell viability decreased as dose of roscovitine increased. The blastocyst rate was significantly higher (proscovitine-treated (20 and 30 μM) groups (48.8, 48.9, 57.9, and 62.9%, respectively) compared to nontreated cyclic cells (20.2%). However, the cleavage rate and total cell number of cloned embryos were similar for all the groups. The number of ICM cells was improved by 30 μM roscovitine treatment (45.25 ± 2.34). The cryosurvival rate of blastocysts derived from cells synchronized with 20 or 30 μM roscovitine was higher compared to that for total confluence group (33.6, 37.8 vs. 23.8%). In conclusion, treatment with 30 μM roscovitine is optimal for harvesting G0/G1 stage cells for producing high quality cloned buffalo embryos, and that it is better than serum-starvation or total confluence for cell synchronization.

  18. Human cloning laws, human dignity and the poverty of the policy making dialogue.

    Science.gov (United States)

    Caulfield, Timothy

    2003-07-29

    The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. The author critiques one of the most commonly used ethical justifications for cloning laws - the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

  19. Human cloning laws, human dignity and the poverty of the policy making dialogue

    Directory of Open Access Journals (Sweden)

    Caulfield Timothy

    2003-07-01

    Full Text Available Abstract Background The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. Discussion The author critiques one of the most commonly used ethical justifications for cloning laws – the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. Summary It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

  20. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  1. Genome-wide uniparental disomy screen in human discarded morphologically abnormal embryos.

    Science.gov (United States)

    Xu, Jiawei; Zhang, Meixiang; Niu, Wenbin; Yao, Guidong; Sun, Bo; Bao, Xiao; Wang, Linlin; Du, Linqing; Sun, Yingpu

    2015-07-21

    Uniparental disomy (UPD) has been shown to be rare in human normal blastocysts, but its frequency in discarded morphologically abnormal embryos and its relevance to embryonic self-correction of aneuploid remains unknown. The aim of this study was to detect UPD in discarded morphologically abnormal embryos. Both discarded morphologically abnormal embryos, including zero-pronuclear zygotes (0PN), one-pronuclear zygotes (1PN), three-pronuclear zygotes (3PN) and 2PN embryos scored as low development potential were cultured into blastocysts then underwent trophectoderm biopsy. Genome-wide UPD screening of the trophectoderm of 241 discarded morphologically abnormal embryo sourced blastocysts showed that UPD occurred in nine embryos. Five embryos exhibited UPDs with euploid chromosomes, and four displayed UPDs with chromosomal aneuploid. The percentage of UPDs among the morphologically abnormal sourced blastocysts was 3.73%, which is significant higher than the percentage observed in normal blastocysts. The frequency of UPD in 3PN-sourced blastocysts was 7.69%, which is significantly higher than that in normal blastocysts. This study provides the first systematic genome-wide profile of UPD in discarded morphologically abnormal embryos. Our results indicated that UPD may be a common phenomenon in discarded morphologically abnormal embryos and may be relevant to human embryonic self-correction.

  2. Researchers and firing squads: questions concerning the use of frozen human embryos.

    Science.gov (United States)

    Tully, Patrick

    2011-10-01

    Is it morally acceptable to use human embryos left over from fertility treatments in research that would harm or destroy them? Many answer "no" to this question on the grounds that all human beings, including human embryos, have a basic moral status that forbids such use. There are some, though, who accept this claim about the basic moral status of human embryos but who believe nevertheless that frozen human embryos which were generated for fertility treatments but which are no longer wanted for that project are a morally acceptable source of human embryonic stem cells and are acceptable subjects of other forms of research that would destroy them in course. The reasoning offered in defense of this position typically employs the claim that since these embryos are going to be discarded anyway, their possibly fruitful use by researchers is a preferable alternative and one that is not inconsistent with their basic moral status. Howard Curzer has offered a well-developed argument of this sort, defending the use of these embryos in the ways mentioned while at the same time allowing for their equal basic moral status. This article challenges Curzer's case and offers reasons to reject the moral acceptability of using even these to-be-discarded embryos as research material.

  3. The ethics of human reproductive cloning: when world views collide.

    Science.gov (United States)

    Cohen, Cynthia B

    2004-01-01

    Two camps in bioethics with seemingly opposing world views have staked out conflicting positions regarding the ethics of human reproductive cloning. These camps do not appear to share common concepts or ways of reasoning through which to exchange views and come to a meeting of minds about uses of this technology. Yet analysis of their respective approaches to several issues surrounding reproductive cloning, such as where the ethical limits of individual reproductive choice lie, whether the use of this technology would violate human dignity, whether it would create risks to the resulting fetuses and children that would make its use intolerable, and whether it would challenge certain core social values, reveals that they are not wholly opposed to one another. Indeed, it displays that they hold certain beliefs, values, and concerns in common. Moreover, it indicates that the different world views that they each presuppose, while flawed in certain respects, do not collide in every respect, but can be reconciled in significant ways that provide fertile ground for agreement about several issues related to human reproductive cloning.

  4. Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos.

    Science.gov (United States)

    Escribá, María-José; Martín, Julio; Rubio, Carmen; Valbuena, Diana; Remohí, José; Pellicer, Antonio; Simón, Carlos

    2006-12-01

    To prove the efficiency of identification and removal of one of the surplus paternal pronuclei in dispermic IVF zygotes to obtain heteroparental blastocysts. Experimental. One hundred fourteen tripronucleated (3PN) embryos from conventional IVF. After informed and signed consent, the patients from Instituto Valenciano Infertilidad (IVI), Valencia, donated their abnormally fertilized embryos. Seventy-two embryos were diploidized by microsurgical removal of the pronucleus located at the farthest position to the second polar body. Forty-two 3PN embryos served as controls. Survival and correction rate; in vitro development up to the blastocyst stage; X, Y, and 18 chromosome determination by triple fluorescent in situ hybridization and, inheritance analysis for 10 polymorphic repeat regions using polymerase chain reaction (PCR) amplification and sequencing. Seventy-eight percent of 3PN zygotes (56/72) survived manipulation and eventually 51 zygotes had two pronuclei (71%). Forty-one percent of manipulated embryos progressed in vitro to the blastocyst stage (21/51). Fluorescent in situ hybridization analysis performed on eight manipulated embryos confirmed their diploid state; all four controls were triploid. Heteroparental inheritances were also confirmed in four of six manipulated embryos. Heteroparental blastocysts can be derived from corrected dispermic zygotes.

  5. Establishment of pregnancies with handmade cloning porcine embryos reconstructed with fibroblasts containing an Alzheimer's disease gene

    DEFF Research Database (Denmark)

    Kragh, P; Li, J; Du, Y

    2008-01-01

    Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, specific genetic manipulations can be performed in donor cells before SCNT to derive pig models for specific human genetic diseases, including the neurodegenerative disorder Alzheimer's disease (AD...... the in vivo developmental capacity, blastocysts were transferred surgically to synchronized recipients. When using PDGF-APPsw-transgenic minipig fibroblasts, the rate of blastocyst formation (mean ± SEM) was 39 ± 3% (164/424). In comparison, non-transgenic fibroblasts resulted in a blastocyst development...

  6. Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2009-01-01

    Summary Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 C/min in a low-temperature (80 C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; 20 C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type. ?? 2009 Cambridge University Press.

  7. Human estrogen sulfotransferase gene (STE): Cloning, structure, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Aksoy, I.A.; Weinshilboum, M. [Mayo Foundation, Rochester, MI (United States)] [and others

    1995-09-01

    Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DBEA) ST gene, STD. The 5{prime} flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5{prime}-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues. 50 refs., 6 figs., 1 tab.

  8. Temporal and Developmental-Stage Variation in the Occurrence of Mitotic Errors in Tripronuclear Human Preimplantation Embryos

    NARCIS (Netherlands)

    Mantikou, Eleni; van Echten-Arends, Jannie; Sikkema-Raddatz, Birgit; van der Veen, Fulco; Repping, Sjoerd; Mastenbroek, Sebastiaan

    2013-01-01

    Mitotic errors during early development of human preimplantation embryos are common, rendering a large proportion of embryos chromosomally mosaic. It is also known that the percentage of diploid cells in human diploid-aneuploid mosaic embryos is higher at the blastocyst than at the cleavage stage. I

  9. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    Directory of Open Access Journals (Sweden)

    Danilo Cimadomo

    2016-01-01

    Full Text Available Preimplantation Genetic Diagnosis and Screening (PGD/PGS for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

  10. The biological basis of non-invasive strategies for selection of human oocytes and embryos.

    Science.gov (United States)

    Scott, Lynette

    2003-01-01

    There is a need for more accurate embryo selection in human assisted reproduction, if the goal of reducing the number of embryos used in embryo transfer is to be realized. Furthermore, any selection strategy should be non-invasive if the embryos are to be used in embryo transfer. Currently, the strategy is selection by one to three parameters in the cleaving- and blastocyst-stage embryo, sometimes with additional pronuclear selection. It is clear that no one system is ideal, as the vast majority of transferred embryos do not implant. As the health of the embryo is largely dictated by the originating gametes, the very early events in oocyte development should be considered. This review will point to the early biological events in the unfertilized and fertilized oocyte that can be scored non-invasively and which can have a profound effect on the later developmental stages. Using a sequential scoring system, with emphasis on the oocyte, a system for selecting the most viable single embryo for transfer may hopefully be achieved.

  11. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    Science.gov (United States)

    Cimadomo, Danilo; Capalbo, Antonio; Ubaldi, Filippo Maria; Scarica, Catello; Palagiano, Antonio; Canipari, Rita; Rienzi, Laura

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential. PMID:26942198

  12. Expression of microRNAs in bovine and human pre-implantation embryo culture media

    Directory of Open Access Journals (Sweden)

    Jenna eKropp

    2014-04-01

    Full Text Available MicroRNAs (miRNA are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, mir-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy.

  13. Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications.

    Science.gov (United States)

    Assou, Said; Boumela, Imène; Haouzi, Delphine; Anahory, Tal; Dechaud, Hervé; De Vos, John; Hamamah, Samir

    2011-01-01

    The first week of human embryonic development comprises a series of events that change highly specialized germ cells into undifferentiated human embryonic stem cells (hESCs) that display an extraordinarily broad developmental potential. The understanding of these events is crucial to the improvement of the success rate of in vitro fertilization. With the emergence of new technologies such as Omics, the gene expression profiling of human oocytes, embryos and hESCs has been performed and generated a flood of data related to the molecular signature of early embryo development. In order to understand the complex genetic network that controls the first week of embryo development, we performed a systematic review and study of this issue. We performed a literature search using PubMed and EMBASE to identify all relevant studies published as original articles in English up to March 2010 (n = 165). We also analyzed the transcriptome of human oocytes, embryos and hESCs. Distinct sets of genes were revealed by comparing the expression profiles of oocytes, embryos on Day 3 and hESCs, which are associated with totipotency, pluripotency and reprogramming properties, respectively. Known components of two signaling pathways (WNT and transforming growth factor-β) were linked to oocyte maturation and early embryonic development. Omics analysis provides tools for understanding the molecular mechanisms and signaling pathways controlling early embryonic development. Furthermore, we discuss the clinical relevance of using a non-invasive molecular approach to embryo selection for the single-embryo transfer program.

  14. Persons and their bodies: how we should think about human embryos.

    Science.gov (United States)

    McLachlan, Hugh V

    2002-01-01

    The status of human embryos is discussed particularly in the light of the claim by Fox, in Health Care Analysis 8 that it would be useful to think of them in terms of cyborg metaphors. It is argued that we should consider human embryos for what they are--partially formed human bodies--rather than for what they are like in some respects (and unlike in others)--cyborgs. However to settle the issue of the status of the embryo is not to answer the moral questions which arise concerning how embryos should be treated. Since persons rather than bodies have rights, embryos do not have rights. However, whether or not embryos have rights, people can have duties concerning them. Furthermore, the persons whose fully developed bodies embryos will, might (or might have) become can have rights. Contrary to what is often assumed, it is not merely persons who have (or have had) living, developed human bodies who have moral rights: so it is argued in this paper.

  15. What justifies the United States ban on federal funding for nonreproductive cloning?

    Science.gov (United States)

    Cunningham, Thomas V

    2013-11-01

    This paper explores how current United States policies for funding nonreproductive cloning are justified and argues against that justification. I show that a common conceptual framework underlies the national prohibition on the use of public funds for cloning research, which I call the simple argument. This argument rests on two premises: that research harming human embryos is unethical and that embryos produced via fertilization are identical to those produced via cloning. In response to the simple argument, I challenge the latter premise. I demonstrate there are important ontological differences between human embryos (produced via fertilization) and clone embryos (produced via cloning). After considering the implications my argument has for the morality of publicly funding cloning for potential therapeutic purposes and potential responses to my position, I conclude that such funding is not only ethically permissible, but also humane national policy.

  16. Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

    Science.gov (United States)

    Wei, Qinzhi; Huang, Haiyan; Yang, Linqing; Yuan, Jianhui; Yang, Xiaohua; Liu, Yungang; Zhuang, Zhixiong

    2014-04-01

    Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type. Copyright © 2012 Wiley Periodicals, Inc.

  17. The Dao of human cloning: utopian/dystopian hype in the British press and popular films.

    Science.gov (United States)

    Jensen, Eric

    2008-04-01

    The issue of human cloning has featured in the national science policy agendas in both the United States and the United Kingdom since the announcement in 1997 of Dolly the cloned sheep's birth in Scotland. Such news stories suggesting the imminent cloning of humans have inspired fictional entertainment media over the years, including numerous popular films. Study 1 examines elite British press coverage of human cloning from 1997 to 2004 (n = 857). Study 2 focuses on five human cloning films released between 1978 and 2003. Two sharply divergent discourses emerged from these data. Unqualified hope and habitually hyped claims of future cures permeated the press discourse. In contrast, the films constructed human cloning as an inherently dangerous technology often wielded by hubristic scientists in the tradition of Frankenstein. Both the predominately positive hype in the broadsheet press and the largely negative hype in the films indicate an impoverished and "thin" public debate on the issue of human cloning.

  18. Cloning and expression of human colon mast cell carboxypeptidase

    Institute of Scientific and Technical Information of China (English)

    Zhang-Quan Chen; Shao-Heng He

    2004-01-01

    AIM: To clone and express the human colon mast cell METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to conrtruct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P.pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli was purified with amylose affinity chromatography and heparin agarose chromatogphy.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.carboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the fonctional study on hMC-CP.

  19. Self-correction in human embryos%胚胎自我修复

    Institute of Scientific and Technical Information of China (English)

    徐艳文

    2013-01-01

    Reanalysis of aneuploid embryos diagnosed by preimplantation genetic screening (PGS) using fluorescence in situ hybridization(FISH) showed that part or all cells in some human cleavage-stage embryos may undergo self-correction during preimplantation development. Putative embryo self-correction mechanisms include embryonic mosaicism, preferential segregation of chromosomal abnormalities to the trophectoderm and extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy. However, embryo self-correction has not been proved in the study using a single nucleotide polymorphism (SNP)microarray-based 24 chromosome aneuploidy screening technology. Neither preferential segregation of aneuploidy to trophectoderm nor uniparental disomy was found. Further study to improve the accuracy of karyotyping on cleavage-stage embryos is definitely needed.

  20. Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

    Directory of Open Access Journals (Sweden)

    Timo Schomann

    2013-01-01

    Full Text Available The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6 of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

  1. Cloning

    Science.gov (United States)

    ... sheep that have been genetically modified to produce milk that contains a human protein essential for blood clotting. The hope is that someday this protein can be purified from the milk and given to humans whose blood does not ...

  2. Demographic profile of states with human cloning laws: morality policy meets political economy.

    Science.gov (United States)

    Stabile, Bonnie

    2007-03-01

    This analysis seeks to identify factors that may shape the policy stance - whether restrictive or permissive - that each state in the United States with a human cloning law in place takes toward human therapeutic cloning. The investigation also considers if cloning policy is more the product of morality politics or political economy. Results show that among states with human cloning policies in place, those with a greater biotechnological capacity, more permissive abortion laws, fewer Evangelical Protestants, and higher political liberalism rankings are more likely to have permissive cloning laws. A higher Roman Catholic population is strongly associated with permissive cloning laws, rather than restrictive cloning laws as originally supposed. Factors with morality policy and economic bases were both found to be associated with cloning policy outcomes. Results suggest that morality policies, though distinct in some ways, do share determinants with public policies based on political economy.

  3. Identification of CD146 Expression in Human and Mouse Preimplantation Embryo

    Institute of Scientific and Technical Information of China (English)

    Hong-bo WANG; Xuan DU; Ya-hui XU; Ze-hua WANG

    2008-01-01

    Objective To investigate whether CD146, a cell adhesion molecule, is expressed in mouse and human preimplantation blastocysts and to localize CD146 in the layer of trophectoderm(TE) and/or inner cell mass(ICM). Methods Human and mouse embryos were collected. Using reverse transcription polymerase chain reaction(RT-PCR), the expression of CD146 mRNA in blastocyst was evaluated in human and mouse embryos. Single embryo immunohistochemical staining was applicated in the examination of the expression of CD146 in protein level. The statistical significance of the data was analyzed using t-test. Results CD146 transcript was detected in all human and mouse preimplantation morula and blastocyst. The expression of CD146 was found to localize in human and mouse compacted morula stage embryos and the TE and ICM of the expanded blastocysts. Conclusion mRNA and protein of CD146 was expressed in preimplantation embryos,which may have a profound influence on early preimplantation development for the differentiation of the trophectoderm and the morphogenesis of the blastocyst.Furthermore, the expression of CD146 in blastocyst stage may be implicated in the assistance of embryo implantation.

  4. Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.

    Directory of Open Access Journals (Sweden)

    Madhuri Salker

    Full Text Available BACKGROUND: Recurrent pregnancy loss (RPL, defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL but increased levels of prokineticin-1 (PROK1, a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered "superfertile", defined by a mean TTP of 3 months or less. CONCLUSIONS: Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

  5. Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

    NARCIS (Netherlands)

    Meng, L.; Wan, Y.; Sun, Y.; Zhang, Y.; Wang, Z.; Song, Y.; Wang, F.

    2013-01-01

    Background - Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos.

  6. My Corporis Fabrica Embryo: An ontology-based 3D spatio-temporal modeling of human embryo development.

    Science.gov (United States)

    Rabattu, Pierre-Yves; Massé, Benoit; Ulliana, Federico; Rousset, Marie-Christine; Rohmer, Damien; Léon, Jean-Claude; Palombi, Olivier

    2015-01-01

    Embryology is a complex morphologic discipline involving a set of entangled mechanisms, sometime difficult to understand and to visualize. Recent computer based techniques ranging from geometrical to physically based modeling are used to assist the visualization and the simulation of virtual humans for numerous domains such as surgical simulation and learning. On the other side, the ontology-based approach applied to knowledge representation is more and more successfully adopted in the life-science domains to formalize biological entities and phenomena, thanks to a declarative approach for expressing and reasoning over symbolic information. 3D models and ontologies are two complementary ways to describe biological entities that remain largely separated. Indeed, while many ontologies providing a unified formalization of anatomy and embryology exist, they remain only descriptive and make the access to anatomical content of complex 3D embryology models and simulations difficult. In this work, we present a novel ontology describing the development of the human embryology deforming 3D models. Beyond describing how organs and structures are composed, our ontology integrates a procedural description of their 3D representations, temporal deformation and relations with respect to their developments. We also created inferences rules to express complex connections between entities. It results in a unified description of both the knowledge of the organs deformation and their 3D representations enabling to visualize dynamically the embryo deformation during the Carnegie stages. Through a simplified ontology, containing representative entities which are linked to spatial position and temporal process information, we illustrate the added-value of such a declarative approach for interactive simulation and visualization of 3D embryos. Combining ontologies and 3D models enables a declarative description of different embryological models that capture the complexity of human

  7. CLONING AND SEQUENCING OF MATURED FRAGMENT OF HUMAN NEVER GROWTH FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    马巍; 吴玲; 王德利; 刘淼; 任惠民; 杨广笑; 王全颖

    2003-01-01

    Objective Molecular cloning and sequencing of the human matured fragment of human nerve growth factor(NGF) gene. Methods Extracting the human genomic DNA from the white blood cells as templates, the gene of NGF was cloned by using PCR and T-vector cloning method. Screening the positive clones and identified by the restriction enzymes, and then the cloned amplified fragment was sequenced and analyzed. Results DNA sequence comparison the cloned gene of NGF with the GenBank (V01511) sequence demonstrated that both of sequences were identical, 354bp length. Conclusion Cloning the NGF gene from the human genomic DNA has paved the way for further study on gene therapy of nerve system injury.

  8. Fresh or frozen? Classifying 'spare' embryos for donation to human embryonic stem cell research.

    Science.gov (United States)

    Ehrich, Kathryn; Williams, Clare; Farsides, Bobbie

    2010-12-01

    United Kingdom (UK) funding to build human embryonic stem cell (hESC) derivation labs within assisted conception units (ACU) was intended to facilitate the 'In-vitro fertilisation (IVF)-stem cell interface', including the flow of fresh 'spare' embryos to stem cell labs. However, in the three sites reported on here, which received this funding, most of the embryos used for hESC research came from long term cryopreservation storage and/or outside clinics. In this paper we explore some of the clinical, technical, social and ethical factors that might help to explain this situation. We report from our qualitative study of the ethical frameworks for approaching women/couples for donation of embryos to stem cell research. Members of staff took part in 44 interviews and six ethics discussion groups held at our study sites between February 2008 and October 2009. We focus here on their articulations of social and ethical, as well as scientific, dimensions in the contingent classification of 'spare' embryos, entailing uncertainty, fluidity and naturalisation in classifying work. Social and ethical factors include acknowledging and responding to uncertainty in classifying embryos; retaining 'fluidity' in the grading system to give embryos 'every chance'; tensions between standardisation and variation in enacting a 'fair' grading system; enhancement of patient choice and control, and prevention of regret; and incorporation of patients' values in construction of ethically acceptable embryo 'spareness' ('frozen' embryos, and embryos determined through preimplantation genetic diagnosis (PGD) to be genetically 'affected'). We argue that the success of the 'built moral environment' of ACU with adjoining stem cell laboratories building projects intended to facilitate the 'IVF-stem cell interface' may depend not only on architecture, but also on the part such social and ethical factors play in configuration of embryos as particular kinds of moral work objects.

  9. Political interventions in U.S. human embryo research: an ethical assessment.

    Science.gov (United States)

    Green, Ronald M

    2010-01-01

    For more than 30 years, beginning with the Reagan administration's refusal to support and provide oversight for embryo research, and continuing to the present in congressionally imposed limits on funding for such research, progress in infertility medicine and the development of stem cell therapies has been seriously delayed by a series of political interventions. In almost all cases, these interventions result from a view of the moral status of human embryo premised largely on religious assumptions. Although some believe that these interventions are valid expressions of religious values in the public sector, it is argued here that they, in fact, contradict Rawls's conception of public reasoning. Both the prohibition of research involving the human embryo as well as bans on federal funding for embryo-related research place the particular religious views of some citizens above the pressing health needs of almost all, and thus violate the ideal of civility implicit in the Rawlsian standard.

  10. X chromosome inactivation is initiated in human preimplantation embryos

    NARCIS (Netherlands)

    van den Berg, Ilse M; Laven, Joop S E; Stevens, Mary; Jonkers, Iris; Galjaard, Robert-Jan; Gribnau, Joost; van Doorninck, J Hikke

    2009-01-01

    X chromosome inactivation (XCI) is the mammalian mechanism that compensates for the difference in gene dosage between XX females and XY males. Genetic and epigenetic regulatory mechanisms induce transcriptional silencing of one X chromosome in female cells. In mouse embryos, XCI is initiated at the

  11. Reproductive cloning and human health: an ethical, international, and nursing perspective.

    Science.gov (United States)

    Sanchez-Sweatman, L R

    2000-03-01

    Human reproductive cloning came to the public's attention when Dolly, a sheep, was cloned in Scotland in 1997. This news quickly spread around the world causing both excitement at the possibilities that cloning techniques could offer, as well as apprehension about the ethical, social and legal implications should human reproductive cloning become possible. Many international organizations, such as the World Health Organization, the International Council of Nurses, and governments were concerned about the impact of human reproductive cloning on human health, dignity and human rights. To this end, many institutions have drafted resolutions, protocols and position statements outlining their concerns. This paper will outline some of the major ethical issues surrounding human reproductive cloning, the position of various international organizations and governments, and specifically the position of the International Council of Nurses.

  12. Cloning and characterization of a functional human ¿-aminobutyric acid (GABA) transporter, human GAT-2

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Meinild, Anne-Kristine; Jensen, Anders A.

    2007-01-01

    Plasma membrane gamma-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human....... The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT......-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable...

  13. Global gene expression profiling of individual human oocytes and embryos demonstrates heterogeneity in early development.

    Directory of Open Access Journals (Sweden)

    Lisa Shaw

    Full Text Available Early development in humans is characterised by low and variable embryonic viability, reflected in low fecundity and high rates of miscarriage, relative to other mammals. Data from assisted reproduction programmes provides additional evidence that this is largely mediated at the level of embryonic competence and is highly heterogeneous among embryos. Understanding the basis of this heterogeneity has important implications in a number of areas including: the regulation of early human development, disorders of pregnancy, assisted reproduction programmes, the long term health of children which may be programmed in early development, and the molecular basis of pluripotency in human stem cell populations. We have therefore investigated global gene expression profiles using polyAPCR amplification and microarray technology applied to individual human oocytes and 4-cell and blastocyst stage embryos. In order to explore the basis of any variability in detail, each developmental stage is replicated in triplicate. Our data show that although transcript profiles are highly stage-specific, within each stage they are relatively variable. We describe expression of a number of gene families and pathways including apoptosis, cell cycle and amino acid metabolism, which are variably expressed and may be reflective of embryonic developmental competence. Overall, our data suggest that heterogeneity in human embryo developmental competence is reflected in global transcript profiles, and that the vast majority of existing human embryo gene expression data based on pooled oocytes and embryos need to be reinterpreted.

  14. Biopsy of human morula-stage embryos: outcome of 215 IVF/ICSI cycles with PGS.

    Directory of Open Access Journals (Sweden)

    Elena E Zakharova

    Full Text Available Preimplantation genetic diagnosis (PGD is commonly performed on biopsies from 6-8-cell-stage embryos or blastocyst trophectoderm obtained on day 3 or 5, respectively. Day 4 human embryos at the morula stage were successfully biopsied. Biopsy was performed on 709 morulae from 215 ICSI cycles with preimplantation genetic screening (PGS, and 3-7 cells were obtained from each embryo. The most common vital aneuploidies (chromosomes X/Y, 21 were screened by fluorescence in situ hybridization (FISH. No aneuploidy was observed in 72.7% of embryos, 91% of those developed to blastocysts. Embryos were transferred on days 5-6. Clinical pregnancy was obtained in 32.8% of cases, and 60 babies were born. Patients who underwent ICSI/PGS treatment were compared with those who underwent standard ICSI treatment by examining the percentage of blastocysts, pregnancy rate, gestational length, birth height and weight. No significant differences in these parameters were observed between the groups. Day 4 biopsy procedure does not adversely affect embryo development in vitro or in vivo. The increased number of cells obtained by biopsy of morulae might facilitate diagnostic screening. There is enough time after biopsy to obtain PGD results for embryo transfer on day 5-6 in the current IVF cycle.

  15. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  16. Ethical attitudes on human cloning among professionals in Taiwan and the policy implications for regulation.

    Science.gov (United States)

    Yang, Che-Ming; Chung, Chun-Chih; Lu, Meei-Shiow; Lin, Chiou-Fen; Chen, Jiun-Shyan

    2005-01-01

    This research focused on understanding the attitudes toward human cloning in Taiwan among professionals in healthcare, law, and religion. The study was conducted utilizing a structured questionnaire. 220 healthcare professionals from two regional hospitals located in Taipei, 351 religious professionals in the northern Taiwan and 711 legal professionals were selected by to receive questionnaires. The valid response rate is 42.1% The questions were generated by an expert panel and represented major arguments in the human cloning debate. There were a total of six Likert scaled questions in the questionnaire. The responses were coded from 1 to 5 with 1 representing strong opposition to human cloning, 3 representing a neutral attitude; and 5 representing a strong favorable attitude toward human cloning. Healthcare professionals had the highest overall average score of 2.14 and the religious professionals had the lowest average at 1.58. All three categories of respondents' attitude toward cloning ranged from mild opposition to strong opposition to human cloning. The religious professionals were more strongly opposed to cloning. Age, education, and religion significantly influenced attitudes toward cloning. Professionals between fifty-one and sixty years old, those with less education, and Roman Catholic professionals were more strongly opposed to cloning. Religious professionals were more strongly opposed to human cloning than professionals in healthcare or law. Younger professionals as an age group demonstrated less opposition to human cloning. Regulation of human cloning will be influenced by professionals in healthcare, law, and religion, and the regulatory environment chosen now will play a pivotal role in influencing the acceptance of human cloning in the future.

  17. Chemical activation with a combination of ionomycin and dehydroleucodine for production of parthenogenetic, ICSI and cloned bovine embryos.

    Science.gov (United States)

    Vichera, G; Alfonso, J; Duque, C C; Silvestre, M A; Pereyra-Bonnet, F; Fernández-Martín, R; Salamone, D

    2010-12-01

    The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μM Io for 4 min followed by 5 μM DhL concentration and after 3-h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6-dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI-embryos or SCNT-embryos is reported here for first time. © 2009 Blackwell Verlag GmbH.

  18. Cloning for therapeutic purposes: ethical and policy considerations.

    Science.gov (United States)

    Hanson, M J

    2001-01-01

    This essay reviews how cloning techniques may be used for therapeutic purposes, analyzes ethical implications, and makes recommendations for public policy discourse. Although cloning may bring many potential benefits, they remain uncertain. Furthermore, human embryo research is morally problematic. Therefore, alternatives to human cloning for therapeutic aims should be sought at present. In addition to central ethical issues, public discourse should maintain an emphasis on the value of the human embryo over scientific expediency, the relativity of health, and the principle of justice. Society should support the laudable mission of medical research, while also attending to the moral concerns often threatened by the promises of scientific progress.

  19. Studies on Construction and Transfer of Cloned Minipig Embryos%小型猪克隆胚的构建及胚胎移植研究

    Institute of Scientific and Technical Information of China (English)

    戴建军; 张德福; 李垚; 顾晓龙; 张廷宇; 吴彩凤; 张树山; 吴斌; 陈会兰; 陈学进

    2011-01-01

    Objective To establish the somatic cell nuclear transfer technology of minipig and provide technical support for studies of transgenic animal models. Methods Using somatic cell nuclear transfer techniques, nuclear transfer was finished with fetal fibroblast cells and ear fibroblast cells (donor cells) from minipig and Fengjing pig respectively. Embryo transfer was carried out using the two kinds of reconstructed embryos. Result There were no significant differences between the two kinds of donor cells in vitro development (P>0.05). Chemical activation after electric fusion could partially improve the blastocyst rates, but the difference was not significant (P>0.05). Four healthy cloned piglets were borned after embryos transfer by the mixture of the two kinds reconstructed embryos. Microsatellite identification of cloned piglets showed whole same with their donor cells in heredity, and had nothing to do with their surrogate sows. Conclusion The method of our study group could be used to produce clone pigs.%目的 建立小型猪的体细胞核移植技术,为开展转基因小型猪动物模型研究提供技术支撑.方法 应用体细胞核移植技术,以小型猪胎儿成纤维细胞和枫泾猪耳成纤维细胞作为供体细胞进行体细胞核移植,并将两种重构胚混合后进行胚胎移植.结果 两种供体细胞在体外发育能力上无显著差异(P>0.05);电融合后进行化学激活能部分提高囊胚发育率,但差异不显著(P>0.05).两种方法生产的小型猪和枫泾猪克隆胚胎混合后,经胚胎移植均能顺利产下4只健康后代.经微卫星亲缘关系鉴定,克隆猪100%与供体细胞相同,与代孕母猪无关.结论 建立的克隆平台能够用于克隆猪的生产.

  20. Entering the Clone Age

    Institute of Scientific and Technical Information of China (English)

    1995-01-01

    Suppose you make your parents so happy,they decide to have another baby just like you.It might be flattering,but how would you feel about having a little brother or sister who is also your twin? A laboratory experiment conducted last fall suggests it may someday be possible.For the first time ever,scientists made exact copies, or clones, of a human embryo.

  1. An in vivo culture system for human embryos using an encapsulation technology: a pilot study

    Science.gov (United States)

    Blockeel, C.; Mock, P.; Verheyen, G.; Bouche, N.; Le Goff, Ph.; Heyman, Y.; Wrenzycki, C.; Höffmann, K.; Niemann, H.; Haentjens, P.; de Los Santos, M.J.; Fernandez-Sanchez, M.; Velasco, M.; Aebischer, P.; Devroey, P.; Simón, C.

    2009-01-01

    BACKGROUND Animal studies have demonstrated better embryo development in vivo than in vitro. This pilot study tested the feasibility of using a novel in utero culture system (IUCS) to obtain normal human fertilization and embryo development. METHODS The IUCS device comprised a perforated silicone hollow tube. The study included 13 patients (<36 years) undergoing a first intracytoplasmic sperm injection (ICSI) treatment and 167 metaphase II oocytes in three groups. In Group 1, 1–2 h after ICSI, sibling oocytes were assigned to IUCS or conventional in vitro culture. The device was retrieved on Day 1, and all zygotes were cultured in vitro till Day 5. In Group 2, fertilized oocytes were assigned on Day 1, embryos retrieved on Day 3 and all embryos cultured till Day 5. In Group 3, after Day 0 assignment, embryos were retrieved on Day 3 for blastomere biopsy and fluorescence in situ hybridization (FISH) and cultured until Day 5. The highest quality blastocysts were transferred on Day 5. RESULTS Fertilization and embryo development were comparable in the in vitro and IUCS arms, with a tendency towards better embryo quality in the IUCS. FISH analysis in Group 3 revealed more normal embryos using the IUCS (P = 0.049). Three clinical pregnancies and live births were obtained: two from the IUCS arm and one from the in vitro arm. CONCLUSIONS Our pilot study shows that this new IUCS appears to be feasible and safe, supporting normal fertilization, embryo development and normal chromosomal segregation. Furthermore, live births are possible after the transient presence of a silicone device in the uterus.Clinicaltrials.gov: NCT00480103. PMID:19273881

  2. Polarity and cell division orientation in the cleavage embryo: from worm to human

    Science.gov (United States)

    Ajduk, Anna; Zernicka-Goetz, Magdalena

    2016-01-01

    Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells—blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos. PMID:26660321

  3. Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis

    Science.gov (United States)

    Vera-Rodriguez, Maria; Chavez, Shawn L.; Rubio, Carmen; Pera, Renee A. Reijo; Simon, Carlos

    2015-01-01

    Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT–qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n=28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature. PMID:26151134

  4. Integrating insulin into single-step culture medium regulates human embryo development in vitro.

    Science.gov (United States)

    Fawzy, Mohamed; Sabry, Mohamed; Nour, Mohamed; Abdelrahman, Mohamed Y; Roshdy, Eman; Magdi, Yasmin; Abdelghafar, Hazem

    2017-02-01

    To evaluate the effect of supplementing single-step embryo culture medium with insulin on human embryo development. Comparative study. Two private centers. The study involved a sibling oocyte split of 5,142 retrieved oocytes from 360 patients. Sibling oocytes split after intracytoplasmic sperm injection for culture from day 0 through day 5 or 6 in insulin-supplemented or control medium. Women were split to receive their embryos from insulin-supplemented or control medium. Clinical pregnancy rate. There were significantly higher rates of clinical, ongoing, and twin pregnancies in the insulin-supplemented arm than in the control arm. On day 3, embryo quality and compaction were higher in insulin-supplemented medium. On day 5, insulin supplementation showed higher rates of blastocyst formation, quality, and cryopreservation. Insulin supplementation of single-step embryo culture medium from day 0 through day 5 or 6 improved clinical pregnancy rate and human embryo development. However, these findings need further confirmation through a multicenter randomized controlled trial that may include other patient populations and different culture media. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Factors affecting the gene expression of in vitro cultured human preimplantation embryos

    NARCIS (Netherlands)

    Mantikou, E.; Jonker, M.J.; Wong, K.M.; van Montfoort, A.P.A.; de Jong, M.; Breit, T.M.; Repping, S.; Mastenbroek, S.

    2016-01-01

    STUDY QUESTION: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? SUMMARY ANSWER: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryo

  6. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Jin Li

    Full Text Available Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf. Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  7. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    Science.gov (United States)

    Li, Jin; Zeng, Guofang; Qi, Yawei; Tang, Xudong; Zhang, Jingjing; Wu, Zeyong; Liang, Jie; Shi, Lei; Liu, Hongwei; Zhang, Peihua

    2015-01-01

    Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  8. Cloning the human gene for macrophage migration inhibitory factor (MIF)

    Energy Technology Data Exchange (ETDEWEB)

    Paralkar, V.; Wistow, G. (National Institutes of Health, Bethesda, MD (United States))

    1994-01-01

    Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. However, recent work strongly suggests a wider role for MIF beyond the immune system. It is expressed specifically in the differentiating cells of the immunologically privileged eye lens and brain, is a delayed early response gene in fibroblasts, and is expressed in many tissues. Here, the authors report the structure of the remarkably small gene for human MIF that has three exons separated by introns of only 189 and 95 bp and covers less than 1 kb. The cloned sequence also includes 1 kb of 5[prime] flanking region. Primer extension and 5[prime] rapid amplification of cDNA ends (RACE) of human brain RNA both indicate the presence of a single transcription start site in a TATA-less promoter. Northern blot analysis shows a single size of MIF mRNA (about 800 nt) in all human tissues examined. In contrast to previous reports, they find no evidence for multiple genes for MIF in the human genome. 20 refs., 3 figs.

  9. Cloning: revisiting an old debate.

    Science.gov (United States)

    Verhey, Allen D

    1994-09-01

    The debate about cloning that took place 25 years ago, although directed toward a different sort of cloning, elucidates fundamental issues currently at stake in reproductive technologies and research. Paul Ramsey and Joseph Fletcher were participants in this early debate. The differences between Ramsey and Fletcher about the meaning and sufficiency of freedom, the understanding and weighing of good and evil, the connection between embodiment and personhood, the relationship of humans with nature, and the meaning of parenthood suggest both a broader agenda for the debate about cloning and a cautious move forward in the development of embryo-splitting.

  10. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    Science.gov (United States)

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  11. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos

    Directory of Open Access Journals (Sweden)

    Daniela Ávila-González

    2015-09-01

    Full Text Available Data from the literature suggest that human embryonic stem cell (hESC lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1 from poor-quality (PQ embryos derived and maintained on human amniotic epithelial cells (hAEC. This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  12. Cloning and characterization of the human PAX7 promoter.

    Science.gov (United States)

    Murmann, O V; Niggli, F; Schäfer, B W

    2000-04-01

    PAX7, a member of the PAX transcription factor gene family, is normally expressed at high levels during development in the neural tube and in skeletal muscle precursor cells. Interestingly, PAX7 expression was also identified in tumor cells developing from these cell types. To date not much is known about the molecular mechanisms controlling the regulation of PAX7 expression. Therefore, we have cloned and sequenced part of the proximal 5'-flanking region of the human PAX7 gene. Computer-based sequence analysis identified putative binding sites for basic transcription factors. Analysis of a series of deletion constructs in different cell types suggested that a distal region containing several E-boxes might be involved in muscle-specific expression of PAX7, and that a distinct proximal region can enhance basal PAX7 expression in tumor cells.

  13. Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

    Science.gov (United States)

    Li, R; Wu, H; Zhuo, W W; Mao, Q F; Lan, H; Zhang, Y; Hua, S

    2015-10-01

    Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.

  14. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    OpenAIRE

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-01-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its o...

  15. Toxicological and melanin synthesis effects of Polygonum multiflorum root extracts on zebrafish embryos and human melanocytes

    Directory of Open Access Journals (Sweden)

    Thanh Thi Hoai Dang

    2016-09-01

    Full Text Available Polygonum multiflorum (PM has been commmonly used as folk medicine for treatment of various conditions, such as early graying of hair in humans. However, there have been limited studies which have evaluated the toxicological and biological effects of PM in vitro as well as in vivo. In this study, PM root extracts in ethyl acetate (PM-E and in distilled water (PM-W were examined for their effects on the development of teratogenic defects/deaths. Additionally, they were evaluated for their effects on melanin formation in human melanocytes and pigmentation in embryos/larvae of wild type strain AB zebrafish (Danio rerio. Our results showed that PM root extracts at concentrations of 40 mg/L and 105 mg/L induced the development of teratogenic defects, including yolk sac edema (or heart edema, hemovascular defects, necrosis and abnormal trunk in zebrafish embryos at 4 days post fertilization; teratogenic indexes (TIs were 1.43 and 0.63 for ethyl acetate extract and distilled water extract, respectively. Our results also demonstrated that PM-W significantly increased the pigmentation level of embryos/larvae and induced melanin formation in human melanocytes. The amount of melanin in PM-W-exposed embryos/larvae was 2.2-fold and 1.71-fold greater than those in the control embryos/larvae and control melanocytes, respectively. Our study also showed that the increased level of pigmentation in PM-W embryos/larvae or melanin biosynthesis in melanocytes were both regulated by activation of tyrosinase. Conclusively, our study suggests that PM root extracts could be used as potential agents for treatment of early hair graying as well as various other diseases related to loss of pigmentation. However, these PM root extracts may also have some negative effects on embryos; therefore it should be careful when using for women during pregnancy. [Biomed Res Ther 2016; 3(9.000: 808-818

  16. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    Directory of Open Access Journals (Sweden)

    Lifeng Liang

    Full Text Available A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH to evaluate accuracy of the results. We found that most (58.1% of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s, partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal

  17. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  18. The origins of genetic variation between individual human oocytes and embryos: implications for infertility.

    Science.gov (United States)

    Delhanty, Joy D A

    2013-12-01

    Human fertility is low in comparison with that seen in other well-studied mammals. The main reason for this state of affairs seems to be the frequent occurrence and persistence of chromosomal errors in the human conceptus. Evidence obtained over the past two decades shows that the exceptionally high incidence of chromosomal anomalies seen in human preimplantation embryos is the result of errors that may occur at various stages during gamete and embryo formation. In rare cases, an error may exist or arise in the premeiotic germ cells; much more commonly it may arise during the first or second meiotic division in the male or female. Highly efficient cell cycle checkpoints in the male ensure that the incidence of aneuploidy in mature sperm is low compared to that in the oocyte. Most 3-day-old embryos created by IVF are chromosomal mosaics, and this persists to a lesser degree to the blastocyst stage on day 5. While aneuploidy of meiotic origin is a major factor affecting the fertility of older women, embryos from most younger women will have predominantly post-zygotic mitotic errors. Couples experiencing RIF are particularly likely to produce highly abnormal (chaotic) embryos by post-zygotic mechanisms.

  19. Cloning and characterization of the human USP22 gene promoter.

    Directory of Open Access Journals (Sweden)

    Jianjun Xiong

    Full Text Available Ubiquitin-specific processing enzyme 22 (USP22 plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5' RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between -210 and -7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (-13 to -7 resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.

  20. Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

    Directory of Open Access Journals (Sweden)

    Abolghasem Esmaeili

    2015-04-01

    Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

  1. Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

    DEFF Research Database (Denmark)

    Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

    2012-01-01

    -points are given as hours after fertilisation Results: The timing of the first two cleavage cycles resulting in a 4-cell embryo was not significantly different between the groups. The timing of the third cleavage cycle, i.e. division to 5, 6, 7 and 8 cells was delayed for embryos cultured in 20% oxygen (P5cell =0......Introduction: Data from a number of studies indicate -but not unequivocally- that culture of embryos in 5% O2 compared to 20% O2 improves blastocyst formation in humans and various animal species and may yield better pregnancy rates in IVF. The detrimental effects of atmospheric oxygen were...... recently demonstrated to occur from first cleavage cycle in mice using time-lapse microscopy, with the largest impact on the pre-compaction stages. However, embryonic development in mice differs in many aspects from human embryonic development. The objective of this retrospective, descriptive study...

  2. Assessment of aneuploidy in human oocytes and preimplantation embryos by chromosome painting

    Energy Technology Data Exchange (ETDEWEB)

    Rougier, N.; Viegas-Pequignot, E.; Plachot, M. [Hospital Necker, Paris (France)] [and others

    1994-09-01

    The poor quality of chromosome preparations often observed after fixation of oocytes and embryos did not usually allow accurate identification of chromosomes involved in non-disjunctions. We, therefore, used chromosome painting to determine the incidence of abnormalities for chromosomes 1 and 7. A total of 50 oocytes inseminated for IVF and showing no signs of fertilization as well as 37 diploid embryos donated for research were fixed according to the Dyban`s technique. Fluorescence in situ hybridization was carried out using whole chromosome painting DNA probes specific for human chromosome 1 and 7. The incidence of aneuploidy was 28%, 10% and 60% for metaphase II, polar body and sperm chromosomes, respectively. The high incidence of aneuploidy observed in sperm prematurely condensed sperm chromosomes is due to the fact that usually far less than 23 sperm chromatids are observed, maybe as a consequence of incomplete chromosome condensation. Thirty seven embryos were analyzed with the same probes. 48% of early embryos were either monosomic 1 or 7 or mosaics comprising blastomeres with 1, 2 or 3 signals. Thus, 8 among the 11 abnormal embryos had hypodiploid cells (25 to 37 chromosomes) indicating either an artefactual loss of chromosomes or a complex anomaly of nuclear division (maltinucleated blastomeres, abnormal migration of chromosomes at anaphase). We therefore calculated a {open_quotes}corrected{close_quotes} incidence of aneuploidy for chromosomes 1 or 7 in early embryos: 18%. 86% of the blastocysts showed mosaicism 2n/3 or 4n as a consequence of the formation of the syncitiotrophoblast. To conclude, chromosome painting is an efficient method to accurately identify chromosomes involved in aneuploidy. This technique should allow us to evaluate the incidence of non-disjunction for all chromosome pairs. Our results confirm the high incidence of chromosome abnormalities occurring as a consequence of meiotic or mitotic non-disjunctions in human oocytes and embryos.

  3. Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

    Directory of Open Access Journals (Sweden)

    Sushil Kumar Mohapatra

    Full Text Available Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT has had a limited applicability due to very low (>1% live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF and Hand-made cloning (TE-HMC, and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

  4. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

    Science.gov (United States)

    Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

    2017-01-01

    CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

  5. Endomorphins fully activate a cloned human mu opioid receptor.

    Science.gov (United States)

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  6. Cloning, expression, and functional analysis of human dopamine D1 receptors

    Institute of Scientific and Technical Information of China (English)

    Wan-chun SUN; Lei JIN; Yan CAO; Li-zhen WANG; Fan MENG; Xing-zu ZHU

    2005-01-01

    Aim: To construct an HEK293 cell line stably expressing human dopamine D1 receptor (D1R). Methods: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D1R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D1R in HEK293 cells was monitored by the [3H]SCH23390 binding assay. The function of D1R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. Results: An HEK293 cell line stably expressing human D1R was obtained. A saturation radioligand binding experiment with [3H]SCH23390 demonstrated that the Kd and Bmax values were 1.5±0.2 nmol/L and 2.94±0.15 nmol/g of protein, respectively. In the[3H]SCH23390 competition assay, D1R agonist SKF38393 displaced[3H]SCH23390 with an IC50 value of 2.0 (1.5-2.8) μmol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D1R expressed in HEK293 cells in a concentration-dependent manner with an EC50 value of 0.25(0.12-0.53) μmol/L and 0.39 (0.27-0.57) μmol/L at 6 h/0.59 (0.22-1.58) μmol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC50 value of 27 (8.6-70) nmol/L.Conclusion: An HEK293 cell line stably expressing human D1R was obtained successfuly. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.

  7. Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection.

    Science.gov (United States)

    Sakkas, D; Percival, G; D'Arcy, Y; Sharif, K; Afnan, M

    2001-12-01

    To determine the most viable embryos for transfer. Study 1: Preselection of early-cleaving 2-cell embryos for transfer. Study 2: Alternating weeks during which preselection was performed and not performed. ART program, Birmingham Women's Hospital, Birmingham, United Kingdom. Patients undergoing IVF or ICSI cycles with transfer on day 2. Culture of all fertilized embryos. Number of fertilized embryos cleaving to the 2-cell stage on day 1, embryo quality, implantation rates, and pregnancy rates. Patients with early-cleaving 2-cell embryos had significantly higher pregnancy and implantation rates (45 of 100 [45.0%] and 58 of 219 [25.5%], respectively) than did patients without early-cleaving 2-cell embryos (31 of 130 [23.8%] and 43 of 290 [14.8%], respectively). In weeks during which preselection was used, the overall pregnancy and implantation rates of the clinic improved. The presence of early-cleaving 2-cell embryos improves a patient's chance of achieving pregnancy. Use of more stringent embryo selection criteria can improve overall pregnancy rates.

  8. Effect of in vitro culture of human embryos on birthweight of newborns

    NARCIS (Netherlands)

    Dumoulin, John C.; Land, Jolande A.; Van Montfoort, Aafke P.; Nelissen, Ewka C.; Coonen, Edith; Derhaag, Josien G.; Schreurs, Inge L.; Dunselman, Gerard A.; Kester, Arnold D.; Geraedts, Joep P.; Evers, Johannes L.

    In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it

  9. Aneuploidy screening of human IVF embryos: Cytogenetic aspects and clinical implications

    NARCIS (Netherlands)

    E.B. Baart (Esther)

    2007-01-01

    textabstractThe introduction of this thesis provides the reader with the necessary background information to understand the rationale behind the studies conducted. It starts with the observation that human incidence of chromosomal abnormalities in oocytes and embryos. Furthermore, an explanation

  10. Effect of in vitro culture of human embryos on birthweight of newborns

    NARCIS (Netherlands)

    Dumoulin, John C.; Land, Jolande A.; Van Montfoort, Aafke P.; Nelissen, Ewka C.; Coonen, Edith; Derhaag, Josien G.; Schreurs, Inge L.; Dunselman, Gerard A.; Kester, Arnold D.; Geraedts, Joep P.; Evers, Johannes L.

    2010-01-01

    In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it

  11. Detection of teratogens in human serum using rat embryo culture: cancer and epilepsy treatments. [Detecting teratogenicity of anticonvulsant and antineoplastic drugs

    Energy Technology Data Exchange (ETDEWEB)

    Chatot, C. L.

    1979-01-01

    Growth (protein and DNA contents) of headfold stage rat embryos cultured for 48 hrs on human serum was enhanced by glucose supplementation. Embryo growth varied with the source of the serum. Sera from 3 of the 19 control subjects produced abnormal embryos. Sera from 5 subjects undergoing cancer chemotherapy and 6 subjects receiving anticonvulsants were either lethal or teratogenic to cultured rat embryos.

  12. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  13. Human serum teratogenicity studies using in vitro cultures of rat embryos

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N.W.; Chatot, C.L.; Plenefisch, J.D.; Carey, S.W.

    1982-01-01

    Those conditions that constitute reproductive risks to man are being analyzed. Particular concern is with those conditions that cannot be or have not been identified by present methodologies. These conditions constitute the majority of factors causing fetal wastages and birth defects. The test system uses intact rat embryos that are cultured in vitro for 2 days. Findings to date suggest that this system may have a number of distinct advantages: (1) whole-embryo culture provides the test with the entire repertoire of processes involved in embryonic development; (2) whole-rat embryos can be cultured on high levels of blood serum; and (3) they can be cultured on serum from human subjects, which provides a direct and unique evaluation of the principal organism of concern. In regard to this last point, it is important to recognize that there is a large range of teratogenic responses and sensitivities to teratogens dependent upon both individual and species differences. (ERB)

  14. No relationship between embryo morphology and successful derivation of human embryonic stem cell lines.

    Directory of Open Access Journals (Sweden)

    Susanne Ström

    Full Text Available BACKGROUND: The large number (30 of permanent human embryonic stem cell (hESC lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002-2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. METHODS: We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. RESULTS: Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. CONCLUSION: Even very poor quality embryos with few cells in the ICM can give origin to hESC lines.

  15. Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

    Science.gov (United States)

    Nagai, Momoko; Minegishi, Katsura; Komada, Munekazu; Tsuchiya, Maiko; Kameda, Tomomi; Yamada, Shigehito

    2016-05-01

    The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies. © 2015 Japanese Teratology Society.

  16. Admixed human embryos and stem cells: legislative, ethical and scientific advances.

    Science.gov (United States)

    Bahadur, G; Iqbal, M; Malik, S; Sanyal, A; Wafa, R; Noble, R

    2008-01-01

    This paper examines the regulatory framework currently governing the creation of animal-human hybrids and chimera embryos in stem cell research, and some of the ethical implications of such research. It discusses the findings of a recent government select committee that considered the topic. It considers the debate around the precise definition of a human embryo, and whether such hybrids therefore fall within the remit of the Human Fertilisation and Embryology Authority. It outlines the advantages of such hybrids, in lessening the need for human egg donors, as well as the moral objections to species boundary violation. It calls for an examination of the scientific benefits of such research to inform debate on the question, and argues for the need to take genuine account of the public's views on this matter.

  17. Cloning, expression and location of RNase9 in human epididymis

    Directory of Open Access Journals (Sweden)

    Lin YQ

    2008-11-01

    Full Text Available Abstract Background Mammalian spermatozoa become fully motile and fertile during transit through the luminal fluid of the epididymis. At least 200 proteins are present in the epididymal lumen, but the potential roles of these luminal proteins in male fertility are unknown. Investigation of the function of these proteins will elucidate the mechanism of sperm maturation, and also provide new drug targets for male contraception. We cloned RNase9 from a human epididymis cDNA library for characterization and analysis of its functions. Findings It was predicted that human RNase9 gene was located on chromosome 14q11.2 and encoded a 205 amino acids protein with a signal peptide of 26 amino acids at the N-terminus. The protein had eight conserved cysteine residues characteristic of the RNase A family members and several potential post-translational modification sites. At the transcriptional level, RNase9 was expressed in a wide variety of tissues, and the expression was higher in men than in boys. RNase9 was localized to the post-equatorial region of the sperms' head. Immunofluorescence staining showed that RNase9 protein was present mostly in the epithelium of the epididymal tubule. Recombinant RNase9 had no ribonuclease activity. In addition, RNase9 had no detectable effect on sperm motility and fertilization as demonstrated by blocking spermatozoa with anti-RNase9 polyclonal serum. Conclusion RNase9 is expressed in a wide variety of tissues. It is located on the post-equatorial region of the sperm head and the epithelium of epididymal tubule. Although RNase9 belongs to the RNase A family, it has no ribonuclease activity.

  18. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  19. Brain stem global gene expression profiles in human spina bifida embryos

    Institute of Scientific and Technical Information of China (English)

    Hong Zhao; Xiang Li; Wan-I Lie; Quanren He; Ting Zhang; Xiaoying Zheng; Ran Zhou; Jun Xie

    2011-01-01

    Environmental and genetic factors influence the occurrence of neural tube defects, such as spina bifida.Specific disease expression patterns will help to elucidate the pathogenesis of disease.However, results obtained from animal models, which often exhibit organism specificity, do not fully explain the mechanisms of human spina bifida onset.In the present study, three embryos with a gestational age of approximately 17 weeks and a confirmed diagnosis of spina bifida, as well as 3 age-matched normal embryos, were obtained from abortions.Fetal brain stem tissues were dissected for RNA isolation, and microarray analyses were conducted to examine profiles of gene expression in brain stems of spina bifida and normal embryos using Affymetrix HG-U1 33A 2.0 GeneChip arrays.Of the 14 500 gene transcripts examined, a total of 182 genes exhibited at least 2.5-fold change in expression, including 140 upregulated and 42 downregulated genes.These genes were placed into 19 main functional categories according to the Gene Ontology Consortium database for biological functions.Of the 182 altered genes, approximately 50% were involved in cellular apoptosis, growth, adhesion, cell cycle, stress, DNA replication and repair, signal transduction, nervous system development, oxidoreduction, immune responses, and regulation of gene transcription.Gene expression in multiple biological pathways was altered in the brain stem of human spina bifida embryos.

  20. The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells

    Directory of Open Access Journals (Sweden)

    Masood Bazrgar

    2016-12-01

    Full Text Available Background: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. Materials and Methods: In this cohort study, we used fluorescence in situ hybridization (FISH to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. Results: Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%, while 2(6.7% were diploid, and 2(6.7% were triploid. Of those with mosaicism, 23(88.5% were determined to be diploid-aneuploid and 3(11.5% were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05; however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. Conclusion: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

  1. Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos.

    Science.gov (United States)

    Mio, Yasuyuki; Maeda, Kazuo

    2008-12-01

    The purpose of this study was to clarify developmental changes of early human embryos by using time-lapse cinematography (TLC). For human ova, fertilization and cleavage, development of the blastocyst, and hatching, as well as consequent changes were repeatedly photographed at intervals of 5-6 days by using an inverse microscope under stabilized temperature and pH. Photographs were taken at 30 frames per second and the movies were studied. Cinematography has increased our understanding of the morphologic mechanisms of fertilization, development, and behavior of early human embryos, and has identified the increased risk of monozygotic twin pregnancy based on prolonged incubation in vitro to the blastocyst stage. Using TLC, we observed the fertilization of an ovum by a single spermatozoon, followed by early cleavages, formation of the morula, blastocyst hatching, changes in the embryonic plates, and the development of monozygotic twins from the incubated blastocysts.

  2. The influence of early embryo traits on human embryonic stem cell derivation efficiency.

    Science.gov (United States)

    O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

    2011-05-01

    Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

  3. Variations and voids: the regulation of human cloning around the world.

    Science.gov (United States)

    Pattinson, Shaun D; Caulfield, Timothy

    2004-12-13

    No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC). While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.

  4. Variations and voids: the regulation of human cloning around the world

    Directory of Open Access Journals (Sweden)

    Caulfield Timothy

    2004-12-01

    Full Text Available Abstract Background No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Methods Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1. We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC and non-reproductive cloning (NRC. Results While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. Conclusions There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.

  5. Just Another Reproductive Technology? The Ethics of Human Reproductive Cloning as an Experimental Medical Procedure

    National Research Council Canada - National Science Library

    D. Elsner

    2006-01-01

    Human reproductive cloning (HRC) has not yet resulted in any live births. There has been widespread condemnation of the practice in both the scientific world and the public sphere, and many countries explicitly outlaw the practice...

  6. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    Science.gov (United States)

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  7. Evaluation of the rat embryo culture system as a predictive test for human teratogens.

    Science.gov (United States)

    Guest, I; Buttar, H S; Smith, S; Varma, D R

    1994-01-01

    Ingestion of the anticonvulsant drug valproic acid and of the angiotensin converting enzyme inhibitor captopril during pregnancy has been associated with abnormal fetal outcome in humans. In contrast, the use of the antiinflammatory drug ibuprofen and the antihistamine diphenhydramine has not been documented to be embryotoxic in humans. We evaluated the rat embryo culture system as a predictive model of teratogenesis, using these four drugs as test agents. Valproic acid, ibuprofen, and diphenhydramine were embryotoxic, inducing concentration-dependent decreases in growth and a significant increase in anomalies. Valproic acid caused an increase in neural tube defects, ibuprofen increased the incidence of abnormal maxillary processes, and diphenhydramine increased the number of embryos with distorted body morphology. These abnormalities were induced at concentrations of valproic acid and diphenhydramine that are used clinically, but ibuprofen only induced toxicity at concentrations greatly exceeding the therapeutic range. Captopril was not embryotoxic up to 5 mM, the highest concentration tested. These results suggest that the rat embryo culture system produces both false positive and false negative data on the teratogenic potential of drugs. Although such an in vitro assay may be suitable to determine the mechanism of teratogenesis, it is not a sensitive indicator of potential human teratogens on its own. These data support the view that in vitro systems can only supplement clinical and epidemiological observations in humans, possibly as a method to determine mechanisms of actions of teratogens.

  8. Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

    Science.gov (United States)

    1987-09-01

    Report No. 4 If MOLECULAR CLONING OF O HUMAN GENE(S) DIRECTING qTHE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASES cc Annual/Final Report 0 N November...62734A I734A875 IAl 451 MOLECULAR CLONING OF HUMAN GEME(S) DIRECTING THE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASE 12. PERSONAL AUTHOR(S) Hermona Soreq...important roles in regulating the pace and mode of function of particular types of synapses. For example, molecular cloning of the nicotinic (44-46) and the

  9. Effect of Adding Human Chorionic Gonadotropin to The Endometrial Preparation Protocol in Frozen Embryo Transfer Cycles

    Directory of Open Access Journals (Sweden)

    Maryam Eftekhar

    2012-01-01

    Full Text Available Background: Human chorionic gonadotropin (HCG, one of the initial embryonic signals, isprobably a major regulator of the embryo-endometrial relationship. This study aims to assess theadvantage of HCG supplementation during the secretory phase of hormonally prepared cycles forthe transfer of cryopreserved-thawed embryos.Materials and Methods: This study was a randomized clinical trial. Infertile women who werecandidates for frozen-thawed embryo transfers entered the study and were divided into two groups,HCG and control. The endometrial preparation method was similar in both groups: all women receivedestradiol valerate (6 mg po per day from the second day of the menstrual cycle and progesteronein oil (100 mg intramuscular (I.M. when the endometrial thickness reached 8 mm. Estradiol andprogesterone were continued until the tenth week of gestation. In the HCG group, patients received anHCG 5000 IU injection on the first day of progesterone administration and the day of embryo transfer.Results: In this study, 130 couples participated: 65 in the HCG group and 65 in the control group.There was no statistically significant difference between groups regarding basic characteristics.Implantation rate, chemical pregnancy, clinical pregnancy, ongoing pregnancy, and abortion rateswere similar in both groups.Conclusion: Although HCG has some advantages in assisted reproductive technology (ARTcycles, our study did not show any benefit of HCG supplementation during the secretory phase offrozen cycles (Registration Number: IRCT201107266420N4.

  10. Nuclear transfer technology in mammalian cloning.

    Science.gov (United States)

    Wolf, D P; Mitalipov, S; Norgren, R B

    2001-01-01

    The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

  11. Cell-free DNA in human follicular fluid as a biomarker of embryo quality.

    Science.gov (United States)

    Scalici, E; Traver, S; Molinari, N; Mullet, T; Monforte, M; Vintejoux, E; Hamamah, S

    2014-12-01

    Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes? CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes. CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated. One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample. At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers. Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8-12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = -0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = -0

  12. Addressing the ethical issues raised by synthetic human entities with embryo-like features

    Science.gov (United States)

    Aach, John; Lunshof, Jeantine; Iyer, Eswar; Church, George M

    2017-01-01

    The "14-day rule" for embryo research stipulates that experiments with intact human embryos must not allow them to develop beyond 14 days or the appearance of the primitive streak. However, recent experiments showing that suitably cultured human pluripotent stem cells can self-organize and recapitulate embryonic features have highlighted difficulties with the 14-day rule and led to calls for its reassessment. Here we argue that these and related experiments raise more foundational issues that cannot be fixed by adjusting the 14-day rule, because the framework underlying the rule cannot adequately describe the ways by which synthetic human entities with embryo-like features (SHEEFs) might develop morally concerning features through altered forms of development. We propose that limits on research with SHEEFs be based as directly as possible on the generation of such features, and recommend that the research and bioethics communities lead a wide-ranging inquiry aimed at mapping out solutions to the ethical problems raised by them. DOI: http://dx.doi.org/10.7554/eLife.20674.001

  13. Jewish points of views on the animation of the human embryo

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    Ks. Artur Aleksiejuk

    2014-11-01

    Full Text Available According to biblical anthropology, human beings are composed of body and soul. The question arises, however, at what moment does the body of the embryo possess a spiritual element? Can the breath of God visit the created and already developing in its own biological rhythm embryo? The key issue here is the moment of animation – the origin of a living being, which is created in the image and likeness of God. This article presents various Jewish points of views on the animation of the human embryo, all of which attempt to determine the exact moment at which the soul is breathed into the human body. Rabbinical authorities distinguish five different moments in this process: conception, the forty first day after conception, the birth of the child, the moment of circumcision and the moment in which the child is able to say “Amen.” The first three mentioned cases have the most supporters. The first refers to the simultaneous animation, while the other theories argue for successive animation.

  14. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. (Univ. of California, San Diego, La Jolla (United States)); McPherson, J.P. (Univ. of California, Irvine (United States)); Evans, G.A. (Salk Inst. for Biological Studies, La Jolla, CA (United States))

    1992-02-15

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCr analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

  15. Estimating limits for natural human embryo mortality [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Gavin E. Jarvis

    2016-12-01

    Full Text Available Natural human embryonic mortality is generally considered to be high. Values of 70% and higher are widely cited. However, it is difficult to determine accurately owing to an absence of direct data quantifying embryo loss between fertilisation and implantation. The best available data for quantifying pregnancy loss come from three published prospective studies (Wilcox, Zinaman and Wang with daily cycle by cycle monitoring of human chorionic gonadotrophin (hCG in women attempting to conceive. Declining conception rates cycle by cycle in these studies indicate that a proportion of the study participants were sub-fertile. Hence, estimates of fecundability and pre-implantation embryo mortality obtained from the whole study cohort will inevitably be biased. This new re-analysis of aggregate data from these studies confirms the impression that discrete fertile and sub-fertile sub-cohorts were present. The proportion of sub-fertile women in the three studies was estimated as 28.1% (Wilcox, 22.8% (Zinaman and 6.0% (Wang. The probability of conceiving an hCG pregnancy (indicating embryo implantation was, respectively, 43.2%, 38.1% and 46.2% among normally fertile women, and 7.6%, 2.5% and 4.7% among sub-fertile women. Pre-implantation loss is impossible to calculate directly from available data although plausible limits can be estimated. Based on this new analysis and a model for evaluating reproductive success and failure it is proposed that a plausible range for normal human embryo and fetal mortality from fertilisation to birth is 40-60%.

  16. Major West Indies MRSA clones in human beings: do they travel with their hosts?

    Science.gov (United States)

    Chroboczek, Tomasz; Boisset, Sandrine; Rasigade, Jean-Philippe; Meugnier, Helene; Akpaka, Patrick E; Nicholson, Alison; Nicolas, Muriel; Olive, Claude; Bes, Michele; Vandenesch, François; Laurent, Frederic; Etienne, Jerome; Tristan, Anne

    2013-01-01

    Descriptions of the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) have seldom been produced in the Caribbean, which is a major tourism destination. Using DNA microarrays and spa typing, we characterized 85 MRSA isolates from human skin and soft-tissue infections from five different islands. In the French West Indies (n = 72), the most frequently isolated clones were the same clones that are specifically isolated from mainland France [Lyon (n = 35) and Geraldine (n = 11) clones], whereas the clones that were most frequently isolated from the other islands (n = 13) corresponded with clones that have a worldwide endemic spread [Vienna/Hungarian/Brazilian (n = 5), Panton Valentine leukocidin-positive USA300 (n = 4), New York/Japan (n = 2), and pediatric (n = 1) clones]. The distribution of the major MRSA clones in the French (Guadeloupe and Martinique) and non-French West Indies (Jamaica, Trinidad, and Tobago) is different, and the clones most closely resemble those found in the home countries of the travelers who visit the islands most frequently. The distribution might be affected by tourist migration, which is specific to each island. © 2013 International Society of Travel Medicine.

  17. A resource-based version of the argument that cloning is an affront to human dignity.

    Science.gov (United States)

    McDougall, R

    2008-04-01

    The claim that human reproductive cloning constitutes an affront to human dignity became a familiar one in 1997 as policymakers and bioethicists responded to the announcement of the birth of Dolly the sheep. Various versions of the argument that reproductive cloning is an affront to human dignity have been made, most focusing on the dignity of the child produced by cloning. However, these arguments tend to be unpersuasive and strongly criticised in the bioethical literature. In this paper I put forward a different argument that reproductive cloning is an affront to human dignity, one that looks beyond the dignity of the child produced. I suggest that allocating funds to such a pursuit can affront human dignity by diverting resources away from those existing people who lack sufficient health to enable them to exercise basic rights and liberties. This version of the argument posits cloning as an affront to human dignity in particular circumstances, rather than claiming the technology as intrinsically inconsistent with human dignity.

  18. Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction.

    Science.gov (United States)

    Illmensee, K; Kaskar, K; Zavos, P M

    2005-12-01

    The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.

  19. Identification of putative human T cell receptor delta complementary DNA clones

    Energy Technology Data Exchange (ETDEWEB)

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  20. [The structure of the developing blood vessels of the neocortical anlage of the human embryo].

    Science.gov (United States)

    Korzhevskiĭ, D E; Omel'chenko, N V; Smirnov, E B; Petrova, E S

    2000-01-01

    Using light and electron microscopy the structure of blood vessels of neocortical anlage of human 7-12 embryos was studied. It was shown that at the early stage of formation of intraorgan vascular network the wall of blood vessels of ventricular zone successively differentiate, which is characterized by the appearance of second layer of cells (pericytes), accumulation of basement membrane components, widening of the zone of contacts between endotheliocytes and establishment of the contacts with bipolar cells of neocortex anlage. The morphological data obtained assist in comprehension of physiological aspects of formation of blood brain barrier and regulation of blood flow in human embryonal neocortex.

  1. Can the skull-spine length predict heart size in human embryos?

    Directory of Open Access Journals (Sweden)

    Maria Aimée Vila Bormey

    2012-11-01

    Full Text Available Background: embryo´s length is a global measure, relatively easy to estimate by trained personnel, and it is interesting to investigate its use as a predictor of the size reached by developing internal organs. Objective: To characterize cardiac development and its relationship to the length in human embryos. Methods: A descriptive, correlational and cross-sectional study was conducted at the University of Medical Sciences of Villa Clara, which included five specimens belonging to the Embrioteca of the Medicine School. The specimens were measured, processed trough paraffin method, transversally sectioned and digitalized with aesteroscopy-attached camera. 3.0 SCOPE PHOTO software was used for the study of the six cardiac variables. With SPSS 13,0 descriptive statistics was performed as well as correlation analysis and lineal regression. Results: In the weeks 6, 7 and 8, cardiac area was of 5,19; 4,66 and 8,02 mm2 and pericardiac area was of 7,11; 6,37 and 10,07 mm2. Anteroposterior cardiac diameter was of 2,33; 2,90 and 3,44 mm and transversally measured it was of 3,03; 2,52 and 3,65 mm. Anteroposterior pericardiac diameter was of 2,66; 3,37 and 3,61 mm and transversally measured it was of 3,35; 2,64 and 3,79 mm. Anteroposterior diameters of the heart and their cavity were significantly correlated to craneo-raquis length and lineal regression equations were obtained, thus allowing the calculation of these variables. Conclusions: The present study provides both, cardiac and pericardiac morphometrical values in human embryos between six and eight weeks. Craneo-raquis length in embryos can predict their cardiac and pericardiac size.

  2. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  3. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    Institute of Scientific and Technical Information of China (English)

    Amjad Riaz; Xiaoyang Zhao; Xiangpeng Dai; Wei Li; Lei Liu; Haifeng Wan; Yang Yu; Liu Wang; Qi Zhou

    2011-01-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem(ES)cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved.Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  4. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  5. Early embryo mortality in natural human reproduction: What the data say [version 2; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Gavin E. Jarvis

    2017-06-01

    Full Text Available How many human embryos die between fertilisation and birth under natural conditions? It is widely accepted that natural human embryo mortality is high, particularly during the first weeks after fertilisation, with total prenatal losses of 70% and higher frequently claimed. However, the first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period, and establishing the fate of embryos before this is challenging. Calculations are additionally hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are used to justify quantitative claims regarding embryo loss: (i a hypothesis published by Roberts & Lowe in The Lancet  is widely cited but has no practical quantitative value; (ii life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii studies that measure human chorionic gonadotrophin (hCG reveal losses in the second week of development and beyond, but not before; and (iv the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig’s data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data concluded that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, natural human embryo mortality is lower than often claimed and widely accepted. Estimates for total prenatal mortality of 70% or higher are exaggerated and not supported by the available data.

  6. Human growth hormone binding and stimulation of insulin biosynthesis in cloned rat insulinoma cells

    DEFF Research Database (Denmark)

    Billestrup, Nils

    1985-01-01

    Binding of 125I labelled human growth hormone to cloned insulin producing RIN-5AH cells is described. Binding was specific for somatotropic hormones since both human and rat growth hormone could compete for binding sites, whereas much higher concentrations of lactogenic hormones were needed...

  7. Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

    Science.gov (United States)

    White, Carlee R; MacDonald, William A; Mann, Mellissa R W

    2016-09-01

    In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

  8. Early embryo mortality in natural human reproduction: What the data say [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Gavin E. Jarvis

    2016-11-01

    Full Text Available It is generally accepted that natural human embryo mortality during pregnancy is high – losses of 70% and higher from fertilisation to birth are frequently claimed. The first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period. Establishing the fate of embryos before this is challenging, and hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are cited to justify quantitative claims regarding embryo loss: (i a hypothesis published by Roberts & Lowe in The Lancet  is widely cited but has no quantitative value; (ii life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii studies that measure human chorionic gonadotrophin (hCG reveal losses in the second week of development and beyond, but not before; and (iv the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig’s data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data suggests that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, it is clear that some published estimates of natural embryo mortality are exaggerated. Although available data do not provide a precise estimate, natural human embryo mortality is lower than is often claimed.

  9. Ethical and policy issues surrounding the donation of cryopreserved and fresh embryos for human embryonic stem cell research.

    Science.gov (United States)

    Cohen, Cynthia B

    2009-06-01

    The use of human embryos in human embryonic stem cell (hESC) research raises significant ethical and policy issues associated with their donation. Recent research conducted in several countries assesses the percent of persons with cryopreserved and fresh supernumerary embryos willing to donate them for research, their reasons for considering this option, and the concerns they raise about its personal import. Such research provides new insights into rising ethical and policy questions associated with embryo donation for hESC research that should be addressed. In response to such questions, it is argued here that consent to the donation of supernumerary embryos for hESC research should be sought in two or three stages, depending on whether fresh or frozen embryos are at issue, in order to provide patients and their partners with sufficient time and information before they make a final decision. In addition, steps should be taken to support the voluntariness of their decisions by having personnel other than the treating reproductive specialist or stem cell investigators solicit their consent. Prospective embryo donors should also be given a choice about the uses to which hESCs derived from their donated embryos will be put in order to honor their ethical convictions and ensure that there are sufficient embryos for this research. The well-being and rights of those who donate embryos for this research require the sort of support and protection that can be provided by an ethical and policy framework that allows hESC investigations to move forward according to standards that are transparent and that resound with public values.

  10. Pluripotent stem cells from cloned human embryos: success at long last.

    Science.gov (United States)

    Trounson, Alan; DeWitt, Natalie D

    2013-06-06

    Recently in Cell, Mitalipov and colleagues report an advance that has eluded scientists for over a decade-the successful derivation of embryonic stem cell lines using somatic cell nuclear transfer, or SCNT (Tachibana et al., 2013).

  11. 胚胎移植方法和受体母猪因素对克隆猪生产效率的影响%Effect of Recipient Status and Embryo Transfer Methods on Production of Cloned Pigs

    Institute of Scientific and Technical Information of China (English)

    卫恒习; 李秋艳; 高凤磊; 李燕; 张守全; 李宁

    2012-01-01

    [Objective] In order to establish an efficient embryo transfer technology and improve the production efficiency of cloned pigs, the present study was investigated different embryo transfer methods, gestational age and synchronization status of recipients on cloning pigs. [Method] Cloned embryos ware transferred with different methods to different status recipient, the delivery rates and the cloning efficiency were compared. The optimal synchronization time of recipient was determined by using co-transfer of different kinds of cloned embryos with similar developmental ability at different development stages. [Result] The method of transferring embryos through oviduct umbrella receive higher delivery rate and cloning efficiency (2.2% vs 0.4%, P<0.01) than the method of oviduct puncture, and insemination after embryos transfer had a negative effect on cloning efficiency (0.6% vs 2.2%, P<0.01). The litter size and cloning efficiency were higher when using sows as recipient than gilts ((5.5±0.7) vs (2.7±0.3), P <0.05 and 3.0% vs 0.8%, P<0.01 respectively). Higher cloning efficiency was found in the group of recipient estrous time posterior of 12-36 h to the embryo activation when compared to the meanwhile and prior of 12-24 h groups (2.0% vs 0.5% vs 0%, P<0.05), and the optimal recipient synchronization time is the time of estrous posterior 24 h to embryo activation, and the cloning efficiency reached 3.0%. [ Conclusion ] An efficient embryos transfer technology was established in pig cloning by transferring embryos through oviduct umbrella and by using natural estrous sows beginning heat posterior 24 h to embryo activation as recipient.%[目的]建立高效的胚胎移植技术体系,以提高克隆猪的生产效率.[方法]比较不同移植方法和不同受体母猪状况的胚胎移植分娩率和克隆猪的出生效率,利用体外发育能力相似的不同品种和发育阶段的克隆胚胎混合移植确定最佳的受体母猪发情同期时间.[结果]

  12. What exactly is an exact copy? And why it matters when trying to ban human reproductive cloning in Australia.

    Science.gov (United States)

    Gogarty, B

    2003-04-01

    This paper examines the current Australian regulatory response to human reproductive cloning. The central consideration is the capacity of the current regulatory regime to effectively deter human cloning efforts. A legislative prohibition on human cloning must be both effective and clear enough to allow researchers to know what practices are acceptable. This paper asks whether the current Australian regime evinces these qualities and suggests that Australia should follow the example set in the UK by the enactment of the Human Reproductive Cloning Act 2001.

  13. Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline.

    Science.gov (United States)

    Da Cunha, Violette; Davies, Mark R; Douarre, Pierre-Emmanuel; Rosinski-Chupin, Isabelle; Margarit, Immaculada; Spinali, Sebastien; Perkins, Tim; Lechat, Pierre; Dmytruk, Nicolas; Sauvage, Elisabeth; Ma, Laurence; Romi, Benedetta; Tichit, Magali; Lopez-Sanchez, Maria-José; Descorps-Declere, Stéphane; Souche, Erika; Buchrieser, Carmen; Trieu-Cuot, Patrick; Moszer, Ivan; Clermont, Dominique; Maione, Domenico; Bouchier, Christiane; McMillan, David J; Parkhill, Julian; Telford, John L; Dougan, Gordan; Walker, Mark J; Holden, Matthew T G; Poyart, Claire; Glaser, Philippe

    2014-08-04

    Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

  14. Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction.

    Science.gov (United States)

    Songsivilai, S; Bye, J M; Marks, J D; Hughes-Jones, N C

    1990-12-01

    Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

  15. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

    Science.gov (United States)

    Miller-Pinsler, Lutfiya; Wells, Peter G

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (pcatalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (pcatalase is a determinant of risk for EtOH embryopathies.

  16. Tetranectin, a plasminogen kringle 4-binding protein. Cloning and gene expression pattern in human colon cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R

    1992-01-01

    BACKGROUND: Tetranectin is a recently discovered protein that binds to kringle 4 region of plasminogen (Clemmensen I, Petersen LC, Kluft C. Eur J Biochem 1986; 156:327. EXPERIMENTAL DESIGN: The mRNA encoding human tetranectin was cloned by using degenerate primers in a reverse transcriptase...... reaction followed by polymerase chain reaction amplification. The resulting polymerase chain reaction product was examined by DNA sequencing and subsequently used as probe for screening a human placental cDNA library. A full length cDNA clone (TET-1) was isolated, characterized, and used for Northern blot...

  17. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  18. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research.

    Science.gov (United States)

    Manninen, Bertha Alvarez

    2008-01-31

    One argument used by detractors of human embryonic stem cell research (hESCR) invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event) as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical operation. Finally, I will end the

  19. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research

    Directory of Open Access Journals (Sweden)

    Manninen Bertha

    2008-01-01

    Full Text Available Abstract One argument used by detractors of human embryonic stem cell research (hESCR invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical

  20. Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

    OpenAIRE

    Kurane, I; Brinton, M.A.; Samson, A L; Ennis, F A

    1991-01-01

    Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone re...

  1. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  2. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  3. Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases the In Vitro Developmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

    Directory of Open Access Journals (Sweden)

    Marcin Samiec

    2015-01-01

    Full Text Available The current research was conducted to explore the in vitro developmental outcome and cytological/molecular quality of porcine nuclear-transferred (NT embryos reconstituted with adult bone marrow-derived mesenchymal stem cells (ABM-MSCs that were epigenetically transformed by treatment with nonspecific inhibitor of histone deacetylases, known as trichostatin A (TSA. The cytological quality of cloned blastocysts was assessed by estimation of the total cells number (TCN and apoptotic index. Their molecular quality was evaluated by real-time PCR-mediated quantification of gene transcripts for pluripotency- and multipotent stemness-related markers (Oct4, Nanog, and Nestin. The morula and blastocyst formation rates of NT embryos derived from ABM-MSCs undergoing TSA treatment were significantly higher than in the TSA-unexposed group. Moreover, the NT blastocysts generated using TSA-treated ABM-MSCs exhibited significantly higher TCN and increased pluripotency extent measured with relative abundance of Oct4 and Nanog mRNAs as compared to the TSA-untreated group. Altogether, the improvements in morula/blastocyst yields and quality of cloned pig embryos seem to arise from enhanced abilities for promotion of correct epigenetic reprogramming of TSA-exposed ABM-MSC nuclei in a cytoplasm of reconstructed oocytes. To our knowledge, we are the first to report the successful production of mammalian high-quality NT blastocysts using TSA-dependent epigenomic modulation of ABM-MSCs.

  4. Use of "excess" human embryos for stem cell research: protecting women's rights and health.

    Science.gov (United States)

    Cohen, C B

    2000-01-01

    Proposed National Institutes of Health guidelines for stem cell research are too narrowly drawn and do not adequately protect the freedom of choice and health of women who donate embryos. They need to be expanded to cover not only the point of embryo donation, but also that of embryo creation. Guidelines are provided to ensure that donors undergoing hyperstimulation and egg retrieval gave voluntary informed consent to the production of embryos that might later prove in excess. A standard for determining when embryos have been overproduced is presented to address the possibility that additional embryos will be created for stem cell research in violation of the guidelines and at risk to women's health.

  5. Prosthetic clone and natural human tooth comparison by speckle interferometry

    Science.gov (United States)

    Slangen, Pierre; Corn, Stephane; Fages, Michel; Raynal, Jacques; Cuisinier, Frederic J. G.

    2010-09-01

    New trends in dental prosthodontic interventions tend to preserve the maximum of "body" structure. With the evolution of CAD-CAM techniques, it is now possible to measure "in mouth" the remaining dental tissues. The prosthetic crown is then designed using this shape on which it will be glued on, and also by taking into account the contact surface of the opposite jaw tooth. Several theories discuss on the glue thickness and formulation, but also on the way to evolve to a more biocompatible crown and also new biomechanical concepts. In order to validate these new concepts and materials, and to study the mechanical properties and mechanical integrity of the prosthesis, high resolution optical measurements of the deformations of the glue and the crown are needed. Samples are two intact premolars extracted for orthodontics reasons. The reference sample has no modifications on the tooth while the second sample tooth is shaped to receive a feldspathic ceramic monoblock crown which will be glued. This crown was manufactured with a chairside CAD-CAM system from an intra-oral optical print. The software allows to realize a nearly perfect clone of the reference sample. The necessary space for the glue is also entered with ideal values. This duplication process yields to obtain two samples with identical anatomy for further processing. The glue joint thickness can also be modified if required. The purpose is to compare the behaviour of a natural tooth and its prosthetic clone manufactured with "biomechanical" concepts. Vertical cut samples have been used to deal with planar object observation, and also to look "inside" the tooth. We have developed a complete apparatus enabling the study of the compressive mechanical behaviour of the concerned tooth by speckle interferometry. Because in plane displacements are of great interest for orthodontic measurements1, an optical fiber in-plane sensitive interferometer has been designed. The fibers are wrapped around piezoelectric

  6. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... self-healing cutaneous leishmaniasis. The T-cell clones expressed CD4, and the alpha chain of the T-cell antigen receptor. GP63 reactive T-cell clones activated by antigen or by immobilized anti-CD3 antibody released relative large amounts of interferon-gamma and no or little interleukin-4, thereby...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...

  7. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  8. Integration of the cytogenetic, genetic, and physical maps of the human genome by FISH mapping of CEPH YAC clones

    Energy Technology Data Exchange (ETDEWEB)

    Bray-Ward, P.; Menninger, J.; Lieman, J. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others

    1996-02-15

    This article discusses the genetic mapping of over 950 yeast artificial chromosome (YAC) clones on human chromosomes. This integration of the cytogenetic, genetic and physical maps of the human genome was accomplished using fluorescence in situ hybridization (FISH) mapping and the CEPH library of YAC clones. 27 refs., 2 figs., 1 tab.

  9. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  10. Cloning, characterization, and mRNA expression analysis of novel human fetal cochlear cDNAs.

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Pol, T.J.R. van de; Duijnhoven, G.C.F. van; Hollander, A.I. den; Caat, J. ten; Limpt, V. van; Brunner, H.G.; Kremer, J.M.J.; Cremers, F.P.M.

    2003-01-01

    To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fift

  11. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  12. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    OpenAIRE

    Danilo Cimadomo; Antonio Capalbo; Filippo Maria Ubaldi; Catello Scarica; Antonio Palagiano; Rita Canipari; Laura Rienzi

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most...

  13. The search for truth and freedom: ethical issues surrounding human cloning and stem cell research.

    Science.gov (United States)

    Bruce, Alex

    2002-02-01

    The reality of cloning and stem cell research has provoked wonder, fear and anger. These developments have the potential fundamentally to alter humanity. But how well informed is the range of views being expressed? Is progress being threatened by understandable but uninformed fears? Or are scientists rushing toward an ethical abyss, so concerned with what they can do that they never stop to ask what they should do? This article identifies some of the fears and hopes surrounding cloning and stem cell research. It aims to provoke ethical debate in evaluating such research.

  14. Relationship between Nucleus Swelling and Development Competence of Bovine Cloned Embryos Reconstructed by Enucleated Oocytes with Serum-starved or Serum-fed Fetal Somatic Cells

    Directory of Open Access Journals (Sweden)

    Mokhamad Fahrudin

    2015-10-01

    Full Text Available This study was conducted to examine the occurrence of nuclear remodeling (nucleus swelling and its effectson the subsequent in vitro development of bovine embryos reconstructed by serum-starved and serum-fed somaticcells. Results from this study demonstrated that all of the reconstructed embryos that received serum-starved andserum-fed somatic cells exhibited condensed-nuclei. More than 90% of the transferred nuclei exhibited nuclearenvelope breakdown and premature chromatin condensation which clearly distinct from an intact nucleus. Therewas no significant difference on the degree of nucleus swelling in SS-NT embryos or SF-NT embryos, indicatingthat either serum-starved or confluent somatic cell lines could be reprogrammed by the recipient cytoplasmenvironments in similar pattern. Although the fusion rate was not significantly different among the groups, theproportion of SS-NT embryos which developed to the 2- to 4-cell stage (89.7% and to the 8- to 16-cell stage (74.7%was significantly higher than that of SF-NT embryos. Whereas, the proportion of reconstructed embryos thatdeveloped to the morula and blastocyst stages were not significantly different among the groups. Results of thesestudies demonstrate that reconstructed embryos, which received either serum-starved or serum-fed confluentsomatic cells, showed similar developmental competence to the blastocyst stage.Keywords: nuclear transplantation technique-somatic cells-nucleus swelling

  15. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Directory of Open Access Journals (Sweden)

    Kalpana Dulal

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV. The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  16. [Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene].

    Science.gov (United States)

    Bukhman, V L; Ninkina, N N; Chumakov, P M; Khilenkova, M A; Samarina, O P

    1987-09-01

    Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.

  17. Clinically failed eggs as a source of normal human embryo stem cells.

    Science.gov (United States)

    De Sousa, Paul A; Gardner, John; Sneddon, Sharon; Pells, Steve; Tye, Britt Jorgensen; Dand, Pawlina; Collins, Daniel M; Stewart, Karen; Shaw, Lisa; Przyborski, Stefan; Cooke, Michael; McLaughlin, K John; Kimber, Susan J; Lieberman, Brian A; Wilmut, Ian; Brison, Daniel R

    2009-05-01

    The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

  18. Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos

    Directory of Open Access Journals (Sweden)

    Tiancheng Liu

    2015-01-01

    Full Text Available Evolutionary developmental biology (EVO-DEVO tries to decode evolutionary constraints on the stages of embryonic development. Two models—the “funnel-like” model and the “hourglass” model—have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA. Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies.

  19. Statistical analysis of clone formation in cultures of human stem cells.

    Science.gov (United States)

    Bochkov, N P; Vinogradova, M S; Volkov, I K; Voronina, E S; Kuleshov, N P

    2011-08-01

    We performed a statistical analysis of clone formation from aneuploid cells (chromosomes 6, 8, 11, X) in cultures of bone marrow-derived human multipotent mesenchymal stromal cells by spontaneous level of aneuploidy at different terms of culturing (from 2 to 19 cell cycles). It was found that the duration of cell cycle increased from 65.6 h at passages 2-3 to 164.5 h at passage 12. The expected ratio of aneuploid cells was calculated using modeled 5, 10, 20 and 30% selective preference in reproduction. The size of samples for detecting 10, 25, and 50% increased level of aneuploidy was calculated. The presented principles for evaluation of aneuploid clone formation may be used to distinguish clones of any abnormal cells.

  20. Moderate ovarian stimulation does not increase the incidence of human embryo chromosomal abnormalities in in vitro fertilization cycles.

    Science.gov (United States)

    Labarta, Elena; Bosch, Ernesto; Alamá, Pilar; Rubio, Carmen; Rodrigo, Lorena; Pellicer, Antonio

    2012-10-01

    A high chromosomal abnormalities rate has been observed in human embryos derived from in vitro fertilization (IVF) treatments. The real incidence in natural cycles has been poorly studied, so whether this frequency may be induced by external factors, such as use of gonadotropins for ovarian stimulation, remains unknown. We conducted a prospective cohort study in a University-affiliated private infertility clinic with a comparison between unstimulated and stimulated ovarian cycles in the same women. Preimplantation genetic screening by fluorescence in situ hybridization was performed in all viable d 3 embryos. The primary objective was to compare the incidence of embryo chromosomal abnormalities in an unstimulated cycle and in an ulterior moderate ovarian stimulated cycle. Secondary outcome measures were embryo quality, blastocyst rate of biopsied embryos, number of normal blastocysts per donor, type of chromosomal abnormalities, and clinical outcome. One hundred eighty-five oocyte donors were initially recruited for the unstimulated cycle, and preimplantation genetic screening could be performed in 51 of them, showing 35.3% of embryo chromosomal abnormalities. Forty-six of them later completed a stimulated cycle. The sperm donor sample was the same for both cycles. The proportion of embryos displaying abnormalities in the unstimulated cycle was 34.8% (16 of 46), whereas it was 40.6% (123 of 303) in the stimulated cycle with risk difference=5.8 [95% confidence interval (CI)=-20.6-9.0], and relative risk=1.17 (95% CI=0.77-1.77) (P=0.45). When an intrasubject comparison was made, the abnormalities rate was 34.8% (95% CI=20.5-49.1) in the unstimulated cycle and 38.2% (95% CI=30.5-45.8) in the stimulated cycle [risk difference=3.4 (95% CI=-17.9-11.2); P=0.64]. No differences were observed for embryo quality and type of chromosomal abnormalities. Moderate ovarian stimulation in young normo-ovulatory women does not significantly increase the embryo aneuploidies rate in in

  1. Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E.

    1991-09-01

    The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

  2. Human body motion tracking based on quantum-inspired immune cloning algorithm

    Science.gov (United States)

    Han, Hong; Yue, Lichuan; Jiao, Licheng; Wu, Xing

    2009-10-01

    In a static monocular camera system, to gain a perfect 3D human body posture is a great challenge for Computer Vision technology now. This paper presented human postures recognition from video sequences using the Quantum-Inspired Immune Cloning Algorithm (QICA). The algorithm included three parts. Firstly, prior knowledge of human beings was used, the key joint points of human could be detected automatically from the human contours and skeletons which could be thinning from the contours; And due to the complexity of human movement, a forecasting mechanism of occlusion joint points was addressed to get optimum 2D key joint points of human body; And then pose estimation recovered by optimizing between the 2D projection of 3D human key joint points and 2D detection key joint points using QICA, which recovered the movement of human body perfectly, because this algorithm could acquire not only the global optimal solution, but the local optimal solution.

  3. Sympathy for the Clone: (PostHuman Identities Enhanced by the ‘Evil Science’ Construct and its Commodifying Practices in Contemporary Clone Fiction

    Directory of Open Access Journals (Sweden)

    Jimena Escudero Pérez

    2014-12-01

    Full Text Available The manipulation of human DNA in the form of eugenic pursuit, cloning, genetic engineering etc., has become a well-established subject in science fiction for decades now. In our days, this thematic trend is probably the most prolific one when inspiring narratives in popular culture and also constitutes the source of much bioethical debate. A common pattern derived from these practices is that they generate dystopian scenarios where a community is oppressed and abused by scientific means thus portraying science and its agents as evil. In the case of clone fiction, the focus of this article, the inhumanity of the oppressive powers enhances the questioned humanity of the clones, a particularly complex and evolving type of character that is often commodified. This paper analyses the "evil science" construction and the semiotics of the human/clone identity it produces as displayed in the cases of Never Let Me Go (Kazuo Ishiguro, 2005, The Island (Dir. Michael Bay, USA, 2005, Moon (Dir. Duncan Jones, UK, 2009 and the TV series Orphan Black (created by Graeme Manson & John Fawcett, Canada, 2013–. The cited examples provide references for typified patterns as well as for the development of both the clone figure and the scientific evilness component.

  4. Livestock Origin for a Human Pandemic Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Spoor, Laura E.; McAdam, Paul R.; Weinert, Lucy A.;

    2013-01-01

    ABSTRACT The importance of livestock as a source of bacterial pathogens with the potential for epidemic spread in human populations is unclear. In recent years, there has been a global increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections of healthy...... of emergent clones of methicillin-resistant Staphylococcus aureus (MRSA) that originated in livestock and switched to humans, followed by host-adaptive evolution and epidemic spread in global human populations. Our findings demonstrate that livestock can act as a reservoir for the emergence of new human...

  5. Cloning, chromosome localization and features of a novel human gene, MATH2

    Indian Academy of Sciences (India)

    Lingchen Guo; Min Jiang; Yushu Ma; Haipeng Cheng; Xiaohua Ni; Yangsheng Jin; Yi Xie; Yumin Mao

    2002-04-01

    We report cloning and some features of a novel human gene, MATH2, which encodes a protein of 337 amino acid residues with a basic helix–loop–helix domain and exhibits 98% similarity to mouse Math2. Results of Northern blot analysis revealed two transcripts of the MATH2 gene of 1.7 kb and 2.4 kb in human brain. We localized MATH2 to chromosome 7 at 7p14–15 by matching with the Human Genome Sequence Database. Human MATH2 and mouse Math2 may have the same functions in the nervous system.

  6. Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  7. Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

    Science.gov (United States)

    Bautsch, W; Emde, M; Kretzschmar, T; Köhl, J; Suckau, D; Bitter-Suermann, D

    1992-06-01

    A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

  8. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, S.; Weinshilboum, R.M. [Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, MN (United States); Brandriff, B.F. [Lawrence Livermore National Lab., CA (United States); Ward, A.; Little, P.F.R. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1995-10-10

    Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5{prime}-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5{prime}-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

  9. Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

    Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

  10. Development of transgenic cloned pig models of skin inflammation by DNA transposon-directed ectopic expression of human β1 and α2 integrin.

    Directory of Open Access Journals (Sweden)

    Nicklas Heine Staunstrup

    Full Text Available Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to

  11. Evaluation of cloned cells, animal model, and ATRA sensitivity of human testicular yolk sac tumor

    Directory of Open Access Journals (Sweden)

    Zhao Junfeng

    2012-03-01

    Full Text Available Abstract The testicular yolk sac tumor (TYST is the most common neoplasm originated from germ cells differentiated abnormally, a major part of pediatric malignant testicular tumors. The present study aimed at developing and validating the in vitro and vivo models of TYST and evaluating the sensitivity of TYST to treatments, by cloning human TYST cells and investigating the histology, ultra-structure, growth kinetics and expression of specific proteins of cloned cells. We found biological characteristics of cloned TYST cells were similar to the yolk sac tumor and differentiated from the columnar to glandular-like or goblet cells-like cells. Chromosomes for tumor identification in each passage met nature of the primary tumor. TYST cells were more sensitive to all-trans-retinoic acid which had significantly inhibitory effects on cell proliferation. Cisplatin induced apoptosis of TYST cells through the activation of p53 expression and down-regulation of Bcl- expression. Thus, we believe that cloned TYST cells and the animal model developed here are useful to understand the molecular mechanism of TYST cells and develop potential therapies for human TYST.

  12. NotI linking clones as a tool for joining physical and genetic maps of the human genome.

    Science.gov (United States)

    Allikmets, R L; Kashuba, V I; Pettersson, B; Gizatullin, R; Lebedeva, T; Kholodnyuk, I D; Bannikov, V M; Petrov, N; Zakharyev, V M; Winberg, G

    1994-01-15

    To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes.

  13. NotL linking clones as a tool for joining physical and genetic maps of the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Allikmets, R.L.; Dean, M.; Modi, W. (DynCorp National Cancer Institute, Frederick, MD (United States)); Kholodnyuk, I.D.; Winberg, G.; Klein, G. (Karolinska Institutet, Stockholm (Sweden)); Pettersson, B.; Uhlen, M. (Royal Institute of Technology, Stockholm (Sweden)); Gizatullin, R.; Bannikov, V.M. (and others)

    1994-01-15

    To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes. 41 refs., 3 figs., 2 tabs.

  14. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Miller-Pinsler, Lutfiya [Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario (Canada); Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada)

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

  15. In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial

    NARCIS (Netherlands)

    Kieslinger, Dorit C.; Hao, Zhenxia; Vergouw, Carlijn G.; Kostelijk, Elisabeth H.; Lambalk, Cornelis B.; Le Gac, Séverine

    2015-01-01

    Objective: To compare the development of human embryos in microfluidic devices with culture in standard microdrop dishes, both under static conditions. Design: Prospective randomized controlled trial. Setting: In vitro fertilization laboratory. Patient(s): One hundred eighteen donated frozen-t

  16. Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer%体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究

    Institute of Scientific and Technical Information of China (English)

    刘凤军; 张玉玲; 杨自军; 陈兴启; 孙达权; 王国华; 张涌

    2008-01-01

    [Objective] The aim of this study is to understand the effects of donor cell type, embryo stage, number and transfer position on the efficiency of goat transgenic clone. [Method] Using somatic cell nuclear transfer technology, the single goat fetal fibroblasts (GFF) and mammary gland epithelial cells (GMGE) harboring human lactoferrin (hLF) gene were transferred to the enucleated oocyte. Reconstructed karyoplast-cytoplast couplets were fused, activated, and cultured in vitro. Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients, and blastocysts were transferred into uterine horn. [Result] The pregnancy rate was similar between GFF and GMGE (oviduct transfer:26.47% vs. 20.00%), and between oviduct transfer and uterine horn transfer (26.47% vs. 25.00%) for GFF group; pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group (40.00% vs. 26.67% and 21.43%). [Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type, embryo stage and transfer position but be done by the number of embryo transferred per recipient. In addition, the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.

  17. Molecular cloning and structural analysis of human norepinephrine transporter gene(NETHG)

    Institute of Scientific and Technical Information of China (English)

    GUOLIHE; LIHUAZHU; 等

    1995-01-01

    A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ-aminobutyric acid transporter(hGAT) and a number of other membrane proteins.

  18. Development of High-Throughput Phenotyping of Metagenomic Clones from the Human Gut Microbiome for Modulation of Eukaryotic Cell Growth▿

    OpenAIRE

    2007-01-01

    Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

  19. Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

    Directory of Open Access Journals (Sweden)

    Amparo Galán

    Full Text Available Blastomere fate and embryonic genome activation (EGA during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM (n = 120, stemness (n = 190 and Trophectoderm (TE (n = 45, were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1, stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT, and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR. The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92 such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2 and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4, as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

  20. Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

    Science.gov (United States)

    Galán, Amparo; Montaner, David; Póo, M. Eugenia; Valbuena, Diana; Ruiz, Verónica; Aguilar, Cristóbal; Dopazo, Joaquín; Simón, Carlos

    2010-01-01

    Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented. PMID:21049019

  1. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, H.D.; Tucker, W.Q.J.; Hort, Y.; Martinson, M.E.; Mayo, G.; Clutterbuck, E.J.; Sanderson, C.J.; Young, I.G.

    1987-10-01

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in lambda phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (approx. 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia.

  2. Culture systems: embryo density.

    Science.gov (United States)

    Reed, Michael L

    2012-01-01

    Embryo density is defined as the embryo-to-volume ratio achieved during in vitro culture; in other words, it is the number of embryos in a defined volume of culture medium. The same density can be achieved by manipulating either the number of embryos in a given volume of medium, or manipulating the volume of the medium for a given number of embryos: for example, a microdrop with five embryos in a 50 μl volume under oil has the same embryo-to-volume ratio (1:10 μl) as a microdrop with one embryo in a 10 μl volume under oil (1:10 μl). Increased embryo density can improve mammalian embryo development in vitro; however, the mechanism(s) responsible for this effect may be different with respect to which method is used to increase embryo density.Standard, flat sterile plastic petri dishes are the most common, traditional platform for embryo culture. Microdrops under a mineral oil overlay can be prepared to control embryo density, but it is critical that dish preparation is consistent, where appropriate techniques are applied to prevent microdrop dehydration during preparation, and results of any data collection are reliable, and repeatable. There are newer dishes available from several manufacturers that are specifically designed for embryo culture; most are readily available for use with human embryos. The concept behind these newer dishes relies on fabrication of conical and smaller volume wells into the dish design, so that embryos rest at the lowest point in the wells, and where putative embryotrophic factors may concentrate.Embryo density is not usually considered by the embryologist as a technique in and of itself; rather, the decision to culture embryos in groups or individually is protocol-driven, and is based more on convenience or the need to collect data on individual embryos. Embryo density can be controlled, and as such, it can be utilized as a simple, yet effective tool to improve in vitro development of human embryos.

  3. Transfer of human frozen-thawed embryos with further cleavage during culture increases pregnancy rates

    Directory of Open Access Journals (Sweden)

    Bharat V Joshi

    2010-01-01

    Full Text Available Aim: To compare the pregnancy rate following transfer of frozen-thawed embryos with or without overnight culture after thawing. Settings and Design: This is a retrospective analysis of frozen-thawed embryo transfer (FET cycles performed between January 2006 and December 2008. Materials and Methods: Out of 518 thaw cycles, 504 resulted in embryo transfers (ETs. Of the total FET cycles, 415 were performed after an overnight culture of embryos (group A; and in 89 cycles, ET was performed within 2 hours of embryo thawing (group B. Statistical Analysis: The data were statistically analyzed using chi-square test. Results: We observed that with FET, women ≤30 years of age had a significantly higher (P=0.003 pregnancy rate (PR=28.9% as compared to women >30 years of age (17.5%. A significantly higher (P<0.001FNx08 pregnancy rate was also observed in women receiving 3 frozen-thawed embryos (29% as compared to those who received less than 3 embryos (10.7%. The difference in PR between group A (PR=24.3% and group B (PR=20.3% was not statistically significant. However, within group A, ET with cleaved embryos showed significantly ( P≤0.01 higher pregnancy rate compared to the uncleaved embryos, depending on the number of cleaved embryos transferred. Conclusion: No significant difference was noticed between FETs made with transfer of embryos with overnight culture and those without culture. However, within the cultured group, transfer of embryos cleaved during overnight culture gave significantly higher PR than transfers without any cleavage.

  4. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  5. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  6. The nuclear mitotic apparatus (NuMA) protein: localization and dynamics in human oocytes, fertilization and early embryos.

    Science.gov (United States)

    Alvarez Sedó, Cristian; Schatten, Heide; Combelles, Catherine M; Rawe, Vanesa Y

    2011-06-01

    The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before localizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distribution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necessary for bipolar spindle organization and human oocyte developmental competence.

  7. Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development.

    Science.gov (United States)

    Park, Hyo-Young; Kim, Eun-Young; Lee, Seung-Eun; Choi, Hyun-Yong; Moon, Jeremiah Jiman; Park, Min-Jee; Son, Yeo-Jin; Lee, Jun-Beom; Jeong, Chang-Jin; Lee, Dong-Sun; Riu, Key-Jung; Park, Se-Pill

    2013-12-01

    Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.

  8. Cloned human neuropeptide Y receptor couples to two different second messenger systems.

    OpenAIRE

    Herzog, H.; Hort, Y J; Ball, H J; Hayes, G; Shine, J; Selbie, L A

    1992-01-01

    Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmacological criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY rece...

  9. Cloning and Identification of A Novel Variant of Human Vascular Endothelial Growth Factor

    Institute of Scientific and Technical Information of China (English)

    GUO; Jianli; QU; Shen

    2001-01-01

    A novel variant of human vascular endothelial growth factor (h'VEGF165) cDNA was amplified by nested PCR method from the HL601 cells and was cloned into a eukaryotic expressing vector pcDNA3 to construct a recombinant plasmid pCD-h'VEGF165. The amplified h'VEGF165cDNA fragment was identified by enzyme digestion and DNA sequencing methods. Also, wild-type hVEGF165 cDNA was obtained, identified and cloned into a eukaryotic expressing vector pcDNA3by using the same methods. The results of DNA sequencing showed that h'VEGF165 cDNA cloned from HL601 was 600 bp in size with 8 % of the base sequence in h'VEGF165 cDNA being changed as compared with the base sequence in the wild-type hVEGF165 cDNA. The results of sequencing of hVEGF165 which was cloned from HL60 by us were consistent with the reports completely.

  10. Noninvasive Metabolomic Profiling of Human Embryo Culture Media Using a Simple Spectroscopy Adjunct to Morphology for Embryo Assessment in in Vitro Fertilization (IVF

    Directory of Open Access Journals (Sweden)

    Jiming Hu

    2013-03-01

    Full Text Available Embryo quality is crucial to the outcome of in vitro fertilization (IVF; however, the ability to precisely distinguish the embryos with higher reproductive potential from others is poor. Morphologic evaluation used to play an important role in assessing embryo quality, but it is somewhat subjective. The culture medium is the immediate environment of the embryos in vitro, and a change of the substances in the culture medium is possibly related to the embryo quality. Thus, the present study aims to determine whether metabolomic profiling of the culture medium using Raman spectroscopy adjunct to morphology correlates with the reproductive potential of embryos in IVF and, thus, to look for a new method of assessing embryo quality. Fifty seven spent media samples were detected by Raman spectroscopy. Combined with embryo morphology scores, we found that embryos in culture media with less than 0.012 of sodium pyruvate and more than −0.00085 phenylalanine have a high reproductive potential, with up to 85.7% accuracy compared with clinical pregnancy. So, sodium pyruvate and phenylalanine in culture medium play an important role in the development of the embryo. Raman spectroscopy is an important tool that provides a new and accurate assessment of higher quality embryos.

  11. Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo.

    Science.gov (United States)

    Padgett, R W; Wozney, J M; Gelbart, W M

    1993-04-01

    The type beta transforming growth factor family is composed of a series of processed, secreted growth factors, several of which have been implicated in important regulatory roles in cell determination, inductive interactions, and tissue differentiation. Among these factors, the sequence of the DPP protein from Drosophila is most similar to two of the vertebrate bone morphogenetic proteins, BMP2 and BMP4. Here we report that the human BMP4 ligand sequences can function in lieu of DPP in Drosophila embryos. We introduced the ligand region from human BMP4 into a genomic fragment of the dpp gene in place of the Drosophila ligand sequences and recovered transgenic flies by P-element transformation. We find that this chimeric dpp-BMP4 transgene can completely rescue the embryonic dorsal-ventral patterning defect of null dpp mutant genotypes. We infer that the chimeric DPP-BMP4 protein can be processed properly and, by analogy with the action of other family members, can activate the endogenous DPP receptor to carry out the events necessary for dorsal-ventral patterning. Our evidence suggests that the DPP-BMP4 signal transduction pathway has been functionally conserved for at least 600 million years.

  12. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  13. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  14. Effect of intrauterine injection of human chorionic gonadotropin before embryo transfer on pregnancy rate: A prospective randomized study.

    Science.gov (United States)

    Mostajeran, Fatemeh; Godazandeh, Farzaneh; Ahmadi, Sayed Mehdi; Movahedi, Minoo; Jabalamelian, Seyed Abolfazl

    2017-01-01

    Human chorionic gonadotropin (hCG) as the most important factor to controlled implantation is one of the early embryonic signals in primates that is secreted by the embryo before its implantation. This study was designed to assess the effects of intrauterine injection of hCG before the embryo transfer in an in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycle on pregnancy rate in infertile patients. This randomized study was done on 100 infertile patients in two groups: intervention group received injection of 700 IU of intrauterine hCG 10 min before embryo transfer and control group did not receive hCG. The pregnancy rate was tested 2 weeks after embryo transfer, and if the pregnancy test was positive, a transvaginal ultrasound was performed 3 weeks later to search for signs of pregnancy, such as the presence of a gestational sac, embryo, and fetal heart rate, and confirmed as successful pregnancy. Pregnancy test was positive in 13 (28.6%) of 46 patients in hCG group and in control group was positive in 6 (12.5%) of 48 patients. The pregnancy rate between hCG group and control group was not significantly different (P = 0.54). The pregnancy rate in hCG group with IVF fertilization was 20.8% and in their controls was 7.4% (P = 0.51). The pregnancy rate in hCG group with ICSI fertilization was 36.4% and in their controls was 19% (P = 0.16). The intrauterine injection of 700 IU of hCG before embryo transfer improved pregnancy rate compared to control group but was not significantly different.

  15. Cloning and expression of a novel human HCUTA cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Copper is one of the most important trace elements to life. Human HCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 of E. coli. The whole opening reading frame of HCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed in E. coli BL21 (DE3).

  16. Using therapeutic cloning to fight human disease: a conundrum or reality?

    Science.gov (United States)

    Hall, Vanessa J; Stojkovic, Petra; Stojkovic, Miodrag

    2006-07-01

    The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.

  17. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  18. Heterogeneity of CD4-Positive Human T-Cell Clones Which Recognize the Surface Protein Antigen of Rickettsia typhi

    Science.gov (United States)

    1989-04-01

    5055 " - PROGRAM PROJECT 1TASK IWORK UNIT ELEMENT NO. NO. / NO. IACC SSI II. TITLE (Incluae Security Clasification ) Heterogeneity o. CD4-Positive...Human T-Cell Clones Which Kecognize the Surface Protein Antigen of Rickettsia typhi 12. PERSONAL AOTU-OR(S) Carl M, Vaidya S, Robbins FM, Ching WM...Heterogeneity of CD4-Positive Human T-Cell Clones Which Recognize the Surface Protein Antigen of Rickettsia typhi MITCHELl CARL,* SUSMA VAIDYA,1 FU-MEEI

  19. My Corporis Fabrica Embryo: An ontology-based 3D spatio-temporal modeling of human embryo development

    OpenAIRE

    Rabattu, Pierre-Yves; Massé, Benoit; Ulliana, Federico; Rousset, Marie-Christine; Rohmer, Damien; Léon, Jean-Claude; Palombi, Olivier

    2015-01-01

    Background Embryology is a complex morphologic discipline involving a set of entangled mechanisms, sometime difficult to understand and to visualize. Recent computer based techniques ranging from geometrical to physically based modeling are used to assist the visualization and the simulation of virtual humans for numerous domains such as surgical simulation and learning. On the other side, the ontology-based approach applied to knowledge representation is more and more successfully adopted in...

  20. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  1. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence.

    Science.gov (United States)

    D'Aiuto, L; Antonacci, R; Marzella, R; Archidiacono, N; Rocchi, M

    1993-11-01

    We have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed.

  2. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))

    1993-11-01

    The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

  3. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  4. Effect of Traditional Chinese Herbs Combined with Low Dose Human Menopausal Gonadotropin Applied in Frozen-thawed Embryo Transfer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To assess embryo implantation rate (IR) and pregnancy rate (PR) in women who received Bushen Wengong Decoction (补肾温宫汤, BSWGD), a Chinese herbal formula, combined with low dose of human menopausal gonadotropin (hMG) prior to frozen-thawed embryo transfer (FET). Methods: A total of 262 subjects (674 transferred embryos) who received FET were analyzed retrospectively. In them,122 women were under 30 years old, 106 between 30-35 years and 32 over 35 years. The 85 subjects with normal ovulation were assigned to Group A, the natural menstruation cycling group, on whom no pre-transfer treatment was applied. The other 177 subjects with abnormal ovulation were assigned to Group B, and subdivided, according to the pre-transfer treatment they received, into three groups, Group B1 (50 cases) received BSWGD, Group B2 (58 cases) received hMG and Group B3 (69 cases) received both BSWGD and low dose hMG. The IR and PR of FET in the four groups were compared, and the effect of the embryo cryotime on PR of FET were compared also. Besides, the influencing factors to FET were analyzed. Results: IR and PR were significantly higher in all age sects of Group B3 than those in Group A, showing significant difference (P< 0.05). IR and PR in subjects in age sects of <30 years and > 35 years in group B3 were signifi cantly higher than those in Group B1 ( P<0.05), but no significant difference was shown in the two parameters between Group B 2 and Group B3 ( P>0.05). PR in the subjects who received embryos with cryo-time of > 200 days was significantly lower than that in those with cryo-time of < 100 days (P<0.05). Embryo cryo-time, endometrial thickness, use of BSWGD and use of hMG were of significance in FET ( P< 0.05).Conclusion: A programmed cycle of BSWGD combined with low dose of hMG could improve the embryo IR and PR of FET. Embryo cryo-time, endometrial thickness, and the use of BSWGD and hMG are of significance for FET.

  5. 水牛徒手克隆胚胎培养及冷冻条件的优化%Optimism of Culture and Cryopreservation of Hand-made Clone (HMC) Embryos

    Institute of Scientific and Technical Information of China (English)

    杨春艳; 庞春英; 郑海英; 黄芬香; 王建; 尚江华; 杨炳壮; 梁贤威

    2012-01-01

    This study was conducted to optimize the culture and cryopreservation conditions for buffalo hand-made clone (HMO embryos. Microdrops (MD) , well of the wells (WOW) , flat surface (FS) were adopted to culture HMC reconstruc ted embryos;and 40% EG, 25% EG (ethylene glycol, EG) + 25% DMSO (dimethylsulphoxide, DMSO) and 20% EG + 20% DMSO + 0.5 mol/L sucrose were adopted to vitrify HMC blastocysts. Furthermore, the efficiency of HMC and traditional somatic cell nuclear transfer (SCNT) were also compared. The results showed that the cleavage rate of embryos cultured in WOW was higher than those cultured on FS (P<0. 05) and in MD(P<0. 01) ;the blastocyst rate of embryos derived from WOW system (40.0%) was also higher than those derived from FS (19.8%) and MD (8. 3%) systems (P<0. 01);the cryo-survival rate of blastocysts vitrified with 20% EG + 20% DMSO + 0. 5 mol/L sucrose was higher than those vitrified with 40% EG (P<0. 01) ;both the fusion rate and blasotycst rate of HMC reconstructed embryos were higher than those of SCNT reconstructed embryos (P<0. 05;P<0. 01) , while the cryotolerances of HMC and SCNT blastocysts were not different from each other. These results indicated that HMC could be an alternative to traditional SCNT in buffalo, WOW was most suitable for culture of HMC embryos, and vitrification of HMC blastocysts with 20% EG+20% DMSO + 0. 5 mol/L sucrose could get a reasonable cryosurvival rate.%本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40%EG(ethylene glycol,EG)、25%EG+ 25%DMSO(dimethylsulphoxide,DMSO)和20% EG+20% DMSO+0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率.结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8

  6. The influence of zygote pronuclear morphology on in vitro human embryo development

    Directory of Open Access Journals (Sweden)

    Lidija Križančić-Bombek

    2007-09-01

    Full Text Available Background: The selection of embryos with largest implantation potential is an important part in assisted reproduction. Besides the embryo or blastocyst morphology, selection criteria such as position and orientation of pronuclei (PN in relation to polar body positioning and the number, size and distribution of nucleolar precursor bodies (NPB have been proposed. In our study, a correlation between PN and NBP morphology with the development of early embryos (day 2 of cultivation and blastocysts (day 5 was investigated.Methods: 653 zygotes from 113 IVF (in vitro fertilization and ICSI (intracytoplasmic sperm injection patients, younger than 40 years, were assessed 18–20 hours post-insemination. Optimal zygotes (Z1 had thouching centrally located PN with equall numbers of alligned NPB. Other zygote types differred from Z1 in having scattered NPB in both PN (Z2 or alligned NPB in one PN (Z3 or in PN beeing distant from one another (Z4. For each zygote type a percentage of normal early embryos and blastocysts was calculated.Results: Among 653 assessed zygotes 21.8 % were Z1; 29.1 % Z2, 34.6 % Z3 and 14.5 % Z4. The percentage of normal early embryos decreased from Z1 to Z4 zygote type (70.4 % vs. 55.3 % vs. 59.7 % vs.45.3 %; p < 0.05 as well as the percentage of developed blastocysts (63.4 % vs. 55.3 % vs. 58.8 % vs. 43.2 %. However, the percentages of optimal blastocysts in the four groups did not differ (11.3 % vs. 11.1 % vs. 8.4 % vs. 6.3 %.Conclusions: Best grade zygotes result in batter early embryo and blastocyst development suggesting that zygote morphology can be used in combination with embryo and/or blastocyst evaluation as a method for embryo selection prior to embryo transfer.

  7. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  8. Innervation of the sinu-atrial node and neighbouring regions in two human embryos.

    Science.gov (United States)

    Orts Llorca, F; Domenech Mateu, J M; Puerta Fonolla, J

    1979-03-01

    In human embryos of 20 to 23 mm (36 to 40 days) it is possible to identify on the right side a nerve that we may call the sinusal, which originates by several roots from the nervus vagus dexter (Figs. 1A, B, D), descending through the right ventrolateral face of the primary trachea and right bronchus (Fig. 2, arrows). Beaded in appearance, it gives a fine anastomotic branch which, passing in front of the arteria pulmonalis dextra, passes to the left side (Figs. 2B, C, D; AN). At this level it gives the large branch for the nodus sinoatrialis which, penetrating through the wall of the superior vena cava, provides a rich innervation for the nodus sinoatrialis which is already in an advanced stage of differentiation (Fig. 3, 2; Cy, D, AN). Afterwards it gives fine branches which, following the atrial fold, are distributed throughout the posterior face of the atrium dextrum (Fig. 3). It increases in diameter and, passing through the angle formed by the right pulmonary veins with the atrium dextrum, reaches the intrapericardial portion of the inferior vena cava in the vicinity of its outlet from the atrium (Fig. 3, arrows). The whole innervation is parasympathetic at the stages studied.

  9. Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

    Institute of Scientific and Technical Information of China (English)

    AI-GUO WANG; TAO XIA; QI-LONG CHU; MING ZHANG; FANG LIU; XUE-MIN CHEN; KE-DI YANG

    2004-01-01

    Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

  10. Timetable for upper eyelid development in staged human embryos and fetuses.

    Science.gov (United States)

    Byun, Tae Ho; Kim, Jeong Tae; Park, Hyoung Woo; Kim, Won Kyu

    2011-05-01

    In this study, we examined the development of the upper eyelids to provide a basic understanding of gross anatomical structures and information relative to mechanisms of congenital anomalies in the upper eyelids. We studied the upper eyelids by external and histological observation in 48 human embryos and in fetuses from 5 to 36 weeks postfertilization. The upper eyelid fold began to develop at Stage 18. Upper and lower eyelids fused from the lateral cantus at Stage 22, and fusion was complete by 9 weeks of development. Mesenchymal condensations forming the orbital part of the orbicularis oculi (OO), tarsal plate, and the eyelashes and their appendages, were first seen at Week 9. Definite muscle structures of the upper eyelid, such as the orbital part of the OO and the levator palpebrae superioris and its aponeurosis, and the Müller's muscle were observed at 12 and 14 weeks, respectively. In addition, orbital septum, arterial arcade and orbital fat pad, and tarsal gland (TG) were apparent at 12, 14, and 18 weeks, respectively. Opening of the palpebral fissure was observed at Week 20. In addition, we defined the directional orientation between the levator aponeurosis and orbital septum and the growth pattern of the TG. Our results will be helpful in understanding the normal development of the upper eyelid and the origins of upper eyelid birth defects.

  11. Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

    Science.gov (United States)

    Mousai, M; Hosseini, S M; Hajian, M; Jafarpour, F; Asgari, V; Forouzanfar, M; Nasr-Esfahani, M H

    2015-10-01

    Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

  12. Molecular Cloning of TSARG6 Gene Related to Apoptosis in Human Spermatogenic Cells

    Institute of Scientific and Technical Information of China (English)

    Gang LIU; Guang-Xiu LU; Xiao-Wei XING

    2004-01-01

    Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accessionNo. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan programand PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns islocated in human chromosome lq13.3. Its protein containing 316 amino acid residues is a new member ofHSP40 protein family because the sequence contains the highly conserved J domain which is present in allDna J-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result ofRT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and thetranscript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene whichprobably inhibited human testis spermatogenesis apoptosis.

  13. Sympathy for the Clone: (Post)Human Identities Enhanced by the ‘Evil Science’ Construct and its Commodifying Practices in Contemporary Clone Fiction

    OpenAIRE

    Jimena Escudero Pérez

    2014-01-01

    The manipulation of human DNA in the form of eugenic pursuit, cloning, genetic engineering etc., has become a well-established subject in science fiction for decades now. In our days, this thematic trend is probably the most prolific one when inspiring narratives in popular culture and also constitutes the source of much bioethical debate. A common pattern derived from these practices is that they generate dystopian scenarios where a community is oppressed and abused by scientific means thus ...

  14. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    Energy Technology Data Exchange (ETDEWEB)

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  15. Timing of human preimplantation embryonic development is confounded by embryo origin

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine Kjær; Sundvall Germeys, Linda Karin M; Erlandsen, M.

    2016-01-01

    embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating...... these results may not be generalized to all infertile women. Not all patient-related factors were investigated. WIDER IMPLICATIONS OF THE FINDINGS Our findings underline the importance of treating embryos as dependent observations and suggest a high risk of patient-based confounding in retrospective studies....... The impact of confounders and the embryo origin needs to be addressed in order to apply appropriate statistical models in observational studies. Furthermore, this observation emphasizes the need for RCTs for evaluating use of time-lapse parameters for embryo selection. STUDY FUNDING/COMPETING INTERESTS...

  16. Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

    DEFF Research Database (Denmark)

    Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

    2014-01-01

    PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p cell...... stage (p cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

  17. Language and values in the human cloning debate: a web-based survey of scientists and Christian fundamentalist pastors.

    Science.gov (United States)

    Weasel, Lisa H; Jensen, Eric

    2005-04-01

    Over the last seven years, a major debate has arisen over whether human cloning should remain legal in the United States. Given that this may be the 'first real global and simultaneous news story on biotechnology' (Einsiedel et al., 2002, p.313), nations around the world have struggled with the implications of this newly viable scientific technology, which is often also referred to as somatic cell nuclear transfer. Since the successful cloning of Dolly the sheep in 1997, and with increasing media attention paid to the likelihood of a successful human reproductive clone coupled with research suggesting the medical potential of therapeutic cloning in humans, members of the scientific community and Christian fundamentalist leaders have become increasingly vocal in the debate over U.S. policy decisions regarding human cloning (Wilmut, 2000). Yet despite a surfeit of public opinion polls and widespread opining in the news media on the topic of human cloning, there have been no empirical studies comparing the views of scientists and Christian fundamentalists in this debate (see Evans, 2002a for a recent study of opinion polls assessing religion and attitudes toward cloning). In order to further investigate the values that underlie scientists' and Christian fundamentalist leader's understanding of human cloning, as well as their differential use of language in communicating about this issue, we conducted an open-ended, exploratory survey of practicing scientists in the field of molecular biology and Christian fundamentalist pastors. We then analyzed the responses from this survey using qualitative discourse analysis. While this was not necessarily a representative sample (in quantitative terms, see Gaskell & Bauer, 2000) of each of the groups and the response rate was limited, this approach was informative in identifying both commonalities between the two groups, such as a focus on ethical concerns about reproductive cloning and the use of scientific terminology, as well

  18. The Clone Factory

    Science.gov (United States)

    Stoddard, Beryl

    2005-01-01

    Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

  19. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a in Plasma.

    Directory of Open Access Journals (Sweden)

    Masayuki Ozawa

    Full Text Available High lipoprotein(a [Lp(a] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a [apo(a], the unique component of Lp(a, is found only in primates and humans, the study of human Lp(a has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT techniques, we produced transgenic miniature pigs expressing human apo(a in the plasma. First, we placed the hemagglutinin (HA-tagged cDNA of human apo(a under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a; one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a. Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL of Lp(a in plasma, and the transgenic apo(a gene was transmitted to the offspring. Thus, we generated a human apo(a-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

  20. Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

    Science.gov (United States)

    2016-04-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  1. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    OpenAIRE

    LIANG, YIN-KU; Ping, Wei; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with t...

  2. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  3. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    Energy Technology Data Exchange (ETDEWEB)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. (F. Hoffmann-La Roche Ltd., Basel (Switzerland))

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  4. Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Zhong; Yongping Li; Shuqi Huang; Bo Ning; Chunyan Zhang; Jianliang Zheng; Guanguang Feng

    2002-01-01

    Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

  5. An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion.

    Science.gov (United States)

    Shutova, Maria V; Surdina, Anastasia V; Ischenko, Dmitry S; Naumov, Vladimir A; Bogomazova, Alexandra N; Vassina, Ekaterina M; Alekseev, Dmitry G; Lagarkova, Maria A; Kiselev, Sergey L

    2016-01-01

    The pluripotency of newly developed human induced pluripotent stem cells (iPSCs) is usually characterized by physiological parameters; i.e., by their ability to maintain the undifferentiated state and to differentiate into derivatives of the 3 germ layers. Nevertheless, a molecular comparison of physiologically normal iPSCs to the "gold standard" of pluripotency, embryonic stem cells (ESCs), often reveals a set of genes with different expression and/or methylation patterns in iPSCs and ESCs. To evaluate the contribution of the reprogramming process, parental cell type, and fortuity in the signature of human iPSCs, we developed a complete isogenic reprogramming system. We performed a genome-wide comparison of the transcriptome and the methylome of human isogenic ESCs, 3 types of ESC-derived somatic cells (fibroblasts, retinal pigment epithelium and neural cells), and 3 pairs of iPSC lines derived from these somatic cells. Our analysis revealed a high input of stochasticity in the iPSC signature that does not retain specific traces of the parental cell type and reprogramming process. We showed that 5 iPSC clones are sufficient to find with 95% confidence at least one iPSC clone indistinguishable from their hypothetical isogenic ESC line. Additionally, on the basis of a small set of genes that are characteristic of all iPSC lines and isogenic ESCs, we formulated an approach of "the best iPSC line" selection and confirmed it on an independent dataset.

  6. Comment on a proposed draft protocol for the European Convention on Biomedicine relating to research on the human embryo and fetus.

    Science.gov (United States)

    Lebech, M M

    1998-10-01

    Judge Christian Byk renders service to the Steering Committee on Bioethics of the Council of Europe (CDBI) by proposing a draft of the protocol destined to fill in a gap in international law on the status of the human embryo. This proposal, printed in a previous issue of the Journal of Medical Ethics deserves nevertheless to be questioned on important points. Is Christian Byk proposing to legalise research on human embryos not only in vitro but also in utero?

  7. Preliminarily functional analysis of a cloned novel human gene ADAM29

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ADAM is a family of type I integral membrane proteins which are characterized by sharing a disintegrin and metalloprotease domain and involved in many important physiological processes such as fertilization, neurogenesis and inflammatory response. A novel human ADAM gene--ADAM29, which was cloned in our laboratory, is exclusively expressed in human testis and contains a potential fusion domain. A full-length cDNA of ADAM29 was obtained by using multiple-step PCR. Phylogenetic tree of known mammalian ADAMs specifically expressed in testis was reconstructed. Polyclonal antiserum was raised by immunizing the rabbits with sub-peptide of ADAM29 (Leu268-Asp374) as immunogen. The result of immunohistochemical test on human testis showed that ADAM29 is expressed in different stages of spermatogenesis and in interstitial cells. ADAM29 may play a certain role in the signal transduction during the maturation of testis-associated cells.

  8. Location and expression of neurotrophin-3 and its receptor in the brain of human embryos during early development

    Institute of Scientific and Technical Information of China (English)

    Jian Li; Yongjie Mi; Dajun Ma

    2008-01-01

    BACKGROUND: Cell culture in vitro trials have demonstrated that neurotrophin-3 (NT-3) can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons.This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration.OBJECTIVE: To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development.DESIGN, TIME AND SETTING: An observation study on cells was performed in the Department of Human Anatomy, Histology and Embryology, Chengdu Medical College in September 2007.MATERIALS: Fifteen specimens of fresh human embryo, aged 6 weeks, were used in this study.METHODS: The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo.MAIN OUTCOME MEASURES: Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo.RESULTS: In the early period (aged 6 weeks) of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle.NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and

  9. Cryopreservation of human oocytes, zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification methods

    Directory of Open Access Journals (Sweden)

    Tahani Al-Azawi

    2013-12-01

    Full Text Available Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.

  10. Beyond the 'embryo question': human embryonic stem cell ethics in the context of biomaterial donation in the UK.

    Science.gov (United States)

    Bahadur, G; Morrison, M; Machin, L

    2010-12-01

    Discussion about the ethics of human embryonic stem cell (ESC) research in the UK tends to be dominated by the divisive and potentially intractable issue of the moral status of the embryo. This can have the effect of silencing or marginalizing other concerns, especially in the context of public engagement with science in this field. One such area of potential public concern is the donation of oocytes and embryos to stem cell research. Contemporary research on the views of donors and potential donors about a wide range of biomaterials, from solid organs to gametes and bone marrow, is reviewed and used to illustrate the range and types of ethical concerns articulated by this important group of stakeholders. Attitudes to donation are found to vary according to the type of tissue being donated or collected, the purpose for which donation is being sought and the nature of the recipient of the donation. Pertinently, attitudes towards donating oocytes are found to differ in some respects from donation of embryos or fetal tissue. The implications of these findings for ensuring ethically robust informed consent and publicly acceptable sourcing of human biomaterials for stem cell research are then considered.

  11. MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF HUMAN INTERFERON ALPHA2b GENE

    Directory of Open Access Journals (Sweden)

    I. Made Artika

    2013-01-01

    Full Text Available Human alpha Interferons (hIFNα have been shown to have antiviral, antiproliferative and immunomodulatory activities. The human interferon alpha2b (hIFNα2b, is one of the human interferon alpha2 sub variants, naturally synthesized as a polypeptide of 188 amino acid residues, the first 23 residues of which represents a signal peptide. In the present study, the hIFNα2b gene was expressed after being fused with Glutathione S-Transferase (GST gene. The hIFNα2b gene was amplified from human genomic DNA by using a pair of specific primers, cloned into an Escherichia coli expression vector and expressed in E. coli cells under the direction of the tac promoter. The expressed protein was purified using a one-step affinity chromatography column containing immobilized gluthatione-bound resin. The purified protein was shown to react specifically with anti-human-interferon-alpha antibody, confirming that the protein was the human interferon alpha molecule. This strategy has the potential to be used as an alternative mean for production of pure human interferon α proteins for therapeutic purposes and for further studies on their molecular characterization and mechanism of action.

  12. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  13. "Thin" property and controversial subject matter: Yanner v. Eaton and property rights in human tissue and embryos.

    Science.gov (United States)

    Moses, Lyria Bennett; Gollan, Nicola

    2013-12-01

    This article examines the definitions of "property" offered by the majority of the High Court of Australia in the case of Yanner v Eaton (1999) 201 CLR 351, which involved a statute giving the Crown "property" in fauna. It argues that the majority judges in that case endorsed a flexible or "thin" conception of property that is consistent with recognition of property in "things" such as excised human tissue and in vitro human embryos, despite the many differences between such "things" and ordinary chattels. A similar flexible conception of property was also an important factor in the United Kingdom case of Yearworth v North Bristol NHS Trust[2010] QB 1.

  14. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    Energy Technology Data Exchange (ETDEWEB)

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. (Agouron Institute, La Jolla, CA (USA))

    1988-07-01

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg){sub 9} repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.

  15. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    Science.gov (United States)

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  16. Physical Characterization of human centromeric regions using transformation-associated recombination cloning technology

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir Larionov, Ph D

    2007-06-05

    A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that can be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii

  17. Isolation of human minisatellite loci detected by synthetic tandem repeat probes: direct comparison with cloned DNA fingerprinting probes.

    Science.gov (United States)

    Armour, J A; Vergnaud, G; Crosier, M; Jeffreys, A J

    1992-08-01

    As a direct comparison with cloned 'DNA fingerprinting' probes, we present the results of screening an ordered array Charomid library for hypervariable human loci using synthetic tandem repeat (STR) probes. By recording the coordinates of positive hybridization signals, the subset of clones within the library detected by each STR probe can be defined, and directly compared with the set of clones detected by naturally occurring (cloned) DNA fingerprinting probes. The STR probes vary in the efficiency of detection of polymorphic minisatellite loci; among the more efficient probes, there is a strong overlap with the sets of clones detected by the DNA fingerprinting probes. Four new polymorphic loci were detected by one or more of the STR probes but not by any of the naturally occurring repeats. Sequence comparisons with the probe(s) used to detect the locus suggest that a relatively poor match, for example 10 out of 14 bases in a limited region of each repeat, is sufficient for the positive detection of tandem repeats in a clone in this type of library screening by hybridization. These results not only provide a detailed evaluation of the usefulness of STR probes in the isolation of highly variable loci, but also suggest strategies for the use of these multi-locus probes in screening libraries for clones from hypervariable loci.

  18. Loss of PAF-like activity from human embryo conditioned media (ECM) following HPLC separation.

    Science.gov (United States)

    Adamson, L M; Hanf, V; Mittmann, S G; Tinneberg, H R

    1992-08-21

    Recently a platelet activating factor (PAF)-like activity has been found in embryo conditioned media (ECM) and consequentially been termed embryo-derived PAF (EPAF). Yet it remains unclear whether the embryo-released molecule is in fact PAF or a PAF precursor or inductor in vivo. In this study we shall show that ECM did not induce platelet aggregation in vitro; however, it was possible to detect PAF-activity using the sensitive splenectomized mouse bioassay (SMB). Following lipid extraction, PAF activity was diminished, and after additional HPLC separation completely lost. We propose that the active fraction of ECM is lipid in nature but that this molecule is not PAF. We would rather suggest that this molecule induces the production of PAF by other cell types in vivo.

  19. TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

    Science.gov (United States)

    Kucab, Jill E; Zwart, Edwin P; van Steeg, Harry; Luijten, Mirjam; Schmeiser, Heinz H; Phillips, David H; Arlt, Volker M

    2016-03-01

    3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.

  20. Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

    Science.gov (United States)

    Baldini, A; Archidiacono, N; Carbone, R; Bolino, A; Shridhar, V; Miller, O J; Miller, D A; Ward, D C; Rocchi, M

    1992-01-01

    We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

  1. Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

    Institute of Scientific and Technical Information of China (English)

    Juan Wang; Weibin Sun; Chun Lu; Guixia Tang

    2005-01-01

    Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2 (hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

  2. Molecular cloning and chromosomal localization of the ADH7 gene encoding human class IV ({sigma}) ADH

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hirokazu; Baraona, E.; Lieber, C.S. [Mount Sinai School of Medicine, Bronx, NY (United States)

    1996-01-15

    The ADH7 gene encoding human Class IV ({sigma}) alcohol dehydrogenase (ADH) was cloned from a Caucasian genomic DNA library and characterized. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of {sigma} ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs. 15 refs., 2 figs., 1 tab.

  3. The impact of laser-assisted hatching on the outcome of frozen human embryo transfer cycles.

    Science.gov (United States)

    Kanyo, Katalin; Zeke, Jozsef; Kriston, Rita; Szücs, Zoltan; Cseh, Sandor; Somoskoi, Bence; Konc, Janos

    2016-10-01

    Biochemical modifications of zona pellucida (ZP) result in zona hardening. Zona hardening (ZH) is induced by several factors such as advancing maternal age, in vitro culture conditions and cryopreservation and adversely effects implantation. The objective of the clinical study was to determine whether or not laser-assisted hatching (LAH) applied on day 3 frozen embryos improves the outcome of frozen embryo transfer (FET) cycles in patients with recurrent implantation failure and/or advanced female age. In total, 413 patients of different ages with recurrent implantation failure (maximum three cycles) were involved into the study. Patients were allocated randomly into LAH and control groups. On the day of FET, after thawing and just before FET, the ZP was thinned using a laser system. In the control group no treatment was applied on frozen embryo before transfer. The main outcome measures were clinical pregnancy rate. Overall, the results indicate a tendency that LAH increased (P = 0.08) clinical pregnancy. However, for patients older than 37 years, LAH increased pregnancy rates significantly (P = 0.03). In the LAH and control groups, the age of patients and the number of transferred embryos influenced pregnancy rates (P = 0.01). For patients older than 37 years, no effect of number of transferred embryos was detected (P = 0.14). The incidence of multiple pregnancies also increased in the LAH group (P = 0.01). In conclusion, in older woman, to overcome the negative effect of zona hardening, LAH could be performed on frozen embryos as a routine strategy before FET in frozen cycles in order to increase the possibility of pregnancy formation.

  4. Cloning, functional expression, and characterization of the human prostaglandin E2 receptor EP2 subtype.

    Science.gov (United States)

    Bastien, L; Sawyer, N; Grygorczyk, R; Metters, K M; Adam, M

    1994-04-22

    A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 > iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.

  5. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, C.; Raftogianis, R.; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States)

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  6. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  7. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A L; Cubellis, M V; Masucci, M T

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis...

  8. Human immunodeficiency type-1 virus (HIV-1) infection in serodiscordant couples (SDCs) does not have an impact on embryo quality or intracytoplasmic sperm injection (ICSI) outcome.

    Science.gov (United States)

    Melo, Marco Antonio Barreto; Meseguer, Marcos; Bellver, José; Remohí, José; Pellicer, Antonio; Garrido, Nicolás

    2008-01-01

    To evaluate the embryo quality in our program for human immunodeficiency type-1 virus (HIV-1) serodiscordant couples (SDCs) with the male infected in comparison with a tubal-factor infertility control group. Retrospective case-control study. Instituto Valenciano de Infertilidad, Valencia, Spain. Thirty SDC and 79 control couples without HIV-1 infection attending for intracytoplasmic sperm injection (ICSI). Only first cycles were considered. Controlled ovarian hyperstimulation and ICSI in both groups; sperm wash, nested polymerase chain reaction (PCR) in semen sample, and capacitation by swim-up after thawing the semen sample in the SDC group; and sperm capacitation by swim-up after thawing the semen sample in the control group. ICSI procedure and embryo characteristics (fertilization, cleavage, embryo morphology, and development) and cycle outcome (ongoing pregnancy and miscarriage rates). Fertilization and cleavage rates were similar between the groups. On days 2 and 3 of embryo development, very similar embryo features were found between the groups. There was no difference in mean number of optimal embryos on day 3. When embryos were cultured up to 5-6 days, a significant increase in embryo blockage was found in the SDC group compared with the control group. The mean number of optimal blastocysts on day 6 was comparable in both groups. No difference was found regarding the number of cryopreserved and transferred embryos or implantation, pregnancy, multiple pregnancy, or miscarriage rates between the groups. HIV-1 infection in SDCs with infected males does not appear to have a significantly negative impact on embryo development or ICSI outcome.

  9. 人胚胎移植后剩余胚胎继续体外培养潜能的研究%Potential development to blastocyst of the surplus embryos from human embryo transfer

    Institute of Scientific and Technical Information of China (English)

    薛侠; 赵皖秋; 张四林; 秦臻; 师娟子

    2011-01-01

    Objective To explore the developmental potential of the surplus embryos from human embryo transfer during IVF - ET cycles. Methods All embryos with non - pronucleus (0PN), a single pronudeus (1PN), a number of pronucleus (≥3PN) and 2 pronudeus delaying development in cleavage stage (2PN) were cultured into blastula by the sequential method. Results ① 314 Surplus embryos were collected and formed 152 blastulas(48.41% ) after the sequential culture, among which 53 (34.87%) were high-quality blastula. ② The embryo grade on Day 3 was related to blastocyst rate. The higher embryo grade, the higher blastula formation (54.39%, 52.39%, 49.61% and 21.62% ); ③ Blastocyst formation rates of embryos in 1PN embryos,0PN embryos and D3 from blastocyst embryos classified Ⅲ had higher rates of blastula formation than D3 from blastocyst embryos classified beyond Ⅲ ( P < 0.05). Conclusion Embryos of level Ⅲ and above in D3 are still opportunities for blastocyst formation. The 0PN, 1PN embryo cleavage embryos developed from the D3 can continue to develop in high - quality, until after the formation of blastocysts, and then a pre- implantation genetic diagnosis. If the karyotype is aneuploid karyotype, then it should be frozen or transplanted first. The measures above can improve the oocyte retrieval in patients with accumulation of a single pregnancy, which can also provide resources for embryonic stem cell research.%目的 探讨IVF新鲜周期中D3可用胚胎移植和冷冻后剩余胚胎继续培养的价值.方法 通过囊胚序贯培养法将无原核(0PN)、单个原核(1PN)、多个原核(≥3PN)和卵裂期发育延缓的2原核(2PN)废弃胚胎培养至囊胚期.结果 ① 314枚剩余胚胎于D5~D7形成152枚囊胚(48.41%),其中53枚为优质囊胚(34.87%); ② 胚胎级别越高,囊胚形成率越高(54.39%、52.39%、49.61%和21.62%); ③ 0PN和1PN卵裂发育而来的D3优质胚胎、2PN卵裂发育来的D3评分为Ⅲ级

  10. Assessment of human embryos by time-lapse videography: A comparison of quantitative and qualitative measures between two independent laboratories.

    Science.gov (United States)

    Liu, Yanhe; Copeland, Christopher; Stevens, Adam; Feenan, Katie; Chapple, Vincent; Myssonski, Kim; Roberts, Peter; Matson, Phillip

    2015-12-01

    A total of 488 Day 3 human embryos with known implantation data from two independent in vitro fertilization laboratories were included for analysis, with 270 from Fertility North (FN) and 218 from Canberra Fertility Centre (CFC). Implanting embryos grew at different rates between FN and CFC as indicated in hours of the time intervals between pronuclear fading and the 4- (13.9 ± 1.1 vs. 14.9 ± 1.8), 5- (25.7 ± 1.9 vs. 28.4 ± 3.7) and 8-cell stages (29.0 ± 3.2 vs. 32.2 ± 4.6), as well as the durations of 2- (10.8 ± 0.8 vs. 11.6 ± 1.1), 3- (0.4 ± 0.5 vs. 0.9 ± 1.2), and 4-cell stages (11.8 ± 1.4 vs. 13.6 ± 2.9), all pqualitative measures including poor conventional morphology, direct cleavage, reverse cleavage and 0.05) or non-implanting embryos (30.4% vs. 38.3%, p>0.05) between FN and CFC. Furthermore, implanting embryos favored lower proportions of the above biological events compared to the non-implanting ones in both laboratories (both pquantitative timing parameters may have reduced inter-laboratory transferability; qualitative measures are independent of cell division timings, with potentially improved inter-laboratory reproducibility. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  11. The parental origin correlates with the karyotype of human embryos developing from tripronuclear zygotes.

    Science.gov (United States)

    Joergensen, Mette Warming; Labouriau, Rodrigo; Hindkjaer, Johnny; Stougaard, Magnus; Kolevraa, Steen; Bolund, Lars; Agerholm, Inge Errebo; Sunde, Lone

    2015-03-01

    It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.

  12. Ethical issues in cloning.

    Science.gov (United States)

    Satris, S

    2000-01-01

    There is great public concern with the ethics of human cloning. This paper briefly examines some of what I identify as pseudo-problems or myths associated with cloning, and some of the more substantial ethical concerns.

  13. Just another reproductive technology? The ethics of human reproductive cloning as an experimental medical procedure.

    Science.gov (United States)

    Elsner, D

    2006-10-01

    Human reproductive cloning (HRC) has not yet resulted in any live births. There has been widespread condemnation of the practice in both the scientific world and the public sphere, and many countries explicitly outlaw the practice. Concerns about the procedure range from uncertainties about its physical safety to questions about the psychological well-being of clones. Yet, key aspects such as the philosophical implications of harm to future entities and a comparison with established reproductive technologies such as in vitro fertilisation (IVF) are often overlooked in discussions about HRC. Furthermore, there are people who are willing to use the technology. Several scientists have been outspoken in their intent to pursue HRC. The importance of concerns about the physical safety of children created by HRC and comparisons with concerns about the safety of IVF are discussed. A model to be used to determine when it is acceptable to use HRC and other new assisted reproductive technologies, balancing reproductive freedom and safety concerns, is proposed. Justifications underpinning potential applications of HRC are discussed, and it is determined that these are highly analogous to rationalisations used to justify IVF treatment. It is concluded that people wishing to conceive using HRC should have a prima facie negative right to do so.

  14. Cloning, structural organization, and chromosomal mapping of the human phenol sulfotransferase STP2 gene

    Energy Technology Data Exchange (ETDEWEB)

    Gaedigk, A.; Beatty, B.G.; Grant, D.M. [Hospital for Sick Children, Toronto, Ontario (Canada)

    1997-03-01

    Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p1.2) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family. 27 refs., 3 figs.

  15. Identification of novel human WW domain-containing proteins by cloning of ligand targets.

    Science.gov (United States)

    Pirozzi, G; McConnell, S J; Uveges, A J; Carter, J M; Sparks, A B; Kay, B K; Fowlkes, D M

    1997-06-06

    A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

  16. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

    OpenAIRE

    1991-01-01

    The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-t...

  17. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand.

    Science.gov (United States)

    Thaipadungpanit, Janjira; Wuthiekanun, Vanaporn; Chierakul, Wirongrong; Smythe, Lee D; Petkanchanapong, Wimol; Limpaiboon, Roongrueng; Apiwatanaporn, Apichat; Slack, Andrew T; Suputtamongkol, Yupin; White, Nicholas J; Feil, Edward J; Day, Nicholas P J; Peacock, Sharon J

    2007-10-31

    A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection. Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.

  18. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand.

    Directory of Open Access Journals (Sweden)

    Janjira Thaipadungpanit

    2007-10-01

    Full Text Available A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown.A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34 accounted for 77 (76% of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01. ST34 represented 17/24 (71% of human isolates from other Thai provinces, and 7/8 (88% rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection.Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.

  19. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

    Science.gov (United States)

    Kong, Xiangyi; Yang, Shuting; Gong, Fei; Lu, Changfu; Zhang, Shuoping; Lu, Guangxiu; Lin, Ge

    2016-01-01

    Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In 10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

  20. Cloning and Expression of Recombinant Human Thymosin in Yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Cao Junxia(曹俊霞); Jin Liji; Duan Yanlong; An Lijia

    2003-01-01

    The gene of human thymosin alpha 1(hT(1)was synthesised according to favorite codons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24E that is N-acetylserine were replaced by hT(1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thymosin α1. And also,the Asn-Gly bond was designed to faciliate isolation of the target protein.The fusion gene was cloned into the expression vector, pPIC/9K. The constructs were transformed into HIS4 mutant strain GS115 by electroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.

  1. Isolation and characterization of cloned cDNAs as encoding human liver chlordecone reductase

    Energy Technology Data Exchange (ETDEWEB)

    Winters, C.J.; Molowa, D.T.; Guzelian, P.S. (Medical College of Virginia, Richmond (USA))

    1990-01-30

    Chlordecone (Kepone), a toxic organochlorine pesticide, undergoes bioreduction to chlorodecone alcohol in human liver. This reaction is controlled by a cytosolic enzyme, chlordecone reductase (CDR), which may be of the aldo-keto reductase family of xenobiotic metabolizing enzymes. To further investigate the primary structure and expression of CDR, the authors screened a library of human liver cDNAs cloned in the expression vector {lambda}gt11 and isolated an 800 bp cDNA that directed synthesis of a fusion protein recognized by polyclonal anti-CDR antibodies. Using this cDNA as a probe, they screened two human liver cDNA libraries and found several 1.2-kb cDNAs which would code for polypeptide with 308 residues (35.8 kDa). However, a similar full-length cDNA, possibly the transcript of a pseudogene, contained an in-frame nonsense codon. The deduced protein sequence of CDR showed 65% similarity to the primary structure of human liver aldehyde reductase and 66% similarity to the inferred protein sequence of rat lens aldose reductase. A search of GenBank revealed significant nucleotide similarity to a cDNA coding for bovine lung prostaglandin f synthase and to a partial cDNA coding for frog lens {rho}-crystallin. RNA from adult but not fetal human liver, and from the human hepatoma cell-line Hep G2, contained major (1.6 kb) and minor (2.8 kb) species hybridizable to a CDR cDNA. The relative amounts of these RNAs varied markedly among nine subjects. From this initial description of the nucleotide sequence for a human carbonyl reductase, they conclude that CDR and several related enzymes are part of a novel multigene family involved in the metabolism of such xenobiotics as chlordecone and possibly endogenous substrates.

  2. Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Briggs, Sharon F; Dominguez, Antonia A; Chavez, Shawn L; Reijo Pera, Renee A

    2015-06-01

    The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

  3. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  4. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  5. Molecular cloning and characterization of the human beta-like globin gene cluster.

    Science.gov (United States)

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  6. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  7. Developments in stem cell research and therapeutic cloning: Islamic ethical positions, a review.

    Science.gov (United States)

    Fadel, Hossam E

    2012-03-01

    Stem cell research is very promising. The use of human embryos has been confronted with objections based on ethical and religious positions. The recent production of reprogrammed adult (induced pluripotent) cells does not - in the opinion of scientists - reduce the need to continue human embryonic stem cell research. So the debate continues. Islam always encouraged scientific research, particularly research directed toward finding cures for human disease. Based on the expectation of potential benefits, Islamic teachings permit and support human embryonic stem cell research. The majority of Muslim scholars also support therapeutic cloning. This permissibility is conditional on the use of supernumerary early pre-embryos which are obtained during infertility treatment in vitro fertilization (IVF) clinics. The early pre-embryos are considered in Islamic jurisprudence as worthy of respect but do not have the full sanctity offered to the embryo after implantation in the uterus and especially after ensoulment. In this paper the Islamic positions regarding human embryonic stem cell research and therapeutic cloning are reviewed in some detail, whereas positions in other religious traditions are mentioned only briefly. The status of human embryonic stem cell research and therapeutic cloning in different countries, including the USA and especially in Muslim countries, is discussed.

  8. High efficiency ex vivo cloning of antigen-specific human effector T cells.

    Directory of Open Access Journals (Sweden)

    Michelle A Neller

    Full Text Available While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

  9. Cloning of NHE-1 gene fragment from human lung cancer cells and construction of its antisense expression vector

    Institute of Scientific and Technical Information of China (English)

    WU Guo-ming; HUANG Gui-jun; QIAN Gui-sheng

    2001-01-01

    To clone the partial sequence of Na+/H+ exchanger-1 (NHE-1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy in vivo. Methods: With use of the upstream and downstream primers containing Bam H I and EcoR I in their 5' ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then directionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel electrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector ofNHE-1, pNHE- 1, was constructed successfully.

  10. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

    Science.gov (United States)

    Theaker, Sarah M.; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J.; Cole, David K.; Peakman, Mark; Sewell, Andrew K.; Dolton, Garry

    2016-01-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  11. Popular Theatre for Science Engagement: Audience Engagement with Human Cloning Following a Production of Caryl Churchill's "A Number"

    Science.gov (United States)

    Donkers, Martina; Orthia, Lindy A.

    2016-01-01

    Research into the role of fiction in engaging people with science is a growing area, but a little studied medium in this respect is "popular theatre," or non-pedagogic theatre that exists primarily as a work of art. This study investigated audience engagement with human cloning issues after seeing a performance of Caryl Churchill's 2002…

  12. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, A A; Sheppard, P O

    2001-01-01

    predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane...

  13. A human CD5+ B cell clone that secretes an idiotype-specific high affinity IgM monoclonal antibody.

    NARCIS (Netherlands)

    R.W.J. van der Heijden (Roger); H. Bunschoten; A.C. Hoek (Aad); J. van Es (Johan); M. Punter; A.D.M.E. Osterhaus (Albert); F.G.C.M. Uytdehaag (Fons)

    1991-01-01

    textabstractWe previously demonstrated the occurrence of a naturally arisen human anti-idiotypic B cell clone, that we transformed with EBV (EBV383). We show evidence that EBV383 not only expresses the CD5 surface Ag, but also contains the 2.7-kb mRNA transcript encoding this protein. In addition, w

  14. 山羊MⅡ期卵母细胞胞质支持异种间克隆胚的着床前发育%Goat MⅡ Ooplasts Support Preimplantation Development of Embryos Cloned from Other Species

    Institute of Scientific and Technical Information of China (English)

    徐旭俊; 刘国辉; 陈建泉; 陈娟; 沙红英; 吴友兵; 张爱民; 成国祥

    2008-01-01

    The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase Ⅱ (MⅡ) oocytes matured in vivo and whole cells derived from adult fibroblastsof several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to formblastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118),bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships betweenfusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MⅡ oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.%研究去核山羊(Capra hircus)体内成熟的MⅡ期卵母细胞与异种成年的哺乳动物(包括山羊、渡尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力.结果显示这些异种体细胞核移植重构胚可以完成着床前发育,并形成囊胚.种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469);亚种问或种问体细胞核移植胚的融合率和囊胚发育率分别为:波尔山羊78.18%(541/692)、33.90%(40/118),牛70.53%(146/207).22.52%(25/111),塔尔羊53.51%(61/114)、5.26%(3/570),熊猫79.82%(1159/1452)、8.35%(75/898),人68

  15. Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

    Science.gov (United States)

    Takaya, Akari; Hirohashi, Yoshihiko; Murai, Aiko; Morita, Rena; Saijo, Hiroshi; Yamamoto, Eri; Kubo, Terufumi; Nakatsugawa, Munehide; Kanaseki, Takayuki; Tsukahara, Tomohide; Tamura, Yasuaki; Takemasa, Ichiro; Kondo, Toru; Sato, Noriyuki; Torigoe, Toshihiko

    2016-01-01

    Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

  16. Gadolinium induced apoptosis of human embryo liver L02 cell line by ROS-mediated AIF pathway

    Institute of Scientific and Technical Information of China (English)

    YE Lihua; SHI Zhe; LIU Huixue; YANG Xiaoda; WANG Kui

    2011-01-01

    Gd3+ complexes have a variety of medical applications. In order to shed light on the mechanism of hepatotoxicity of Gd3+ compounds, we investigated the effects of GdCl3 on human embryo liver cell strand (L02 cells). The experimental results showed that long-time exposure to GdC13 resulted in L02 cell apoptosis. The incubation of L02 cells with GdCl3 first induced increase in cellular reactive oxygen species (ROS) and decrease in mitochondrial inner membrane potential (△Ψm). It later resulted in the activation of poly (ADP-ribose) polymerase (PARP) and the release of mitochondrial apoptosis-inducing factor (AIF). The activation of caspase 3, however, was not observed.Antioxidants could significantly reduce GdCl3-induced decrease of △Ψm, release of AIF, and cell apoptosis. Although GdCl3 caused a significant increase in cell membrane permeability in L02, the change of cell membrane permeability was unlikely to be involved in GdCl3-induced cell apoptosis. Overall, our experimental results suggested that GdCl3 induced apoptosis of human embryo liver L02 cell line by ROS-mediated AIF pathway.

  17. Cloning, tissue expression pattern, and chromosome localization of human protein kinase Bγ gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkb? was used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3'-terminal of this fragment was extended to 2788 bp by 'electronic walking' screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBγ and γ, respectively, and was designated as human PKB?. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the highest expression level in brain, and lower levels with variation in the other tissues. By RH mapping, the PKBγ was placed on chromosome 1q43, between markers D1S304 and D1S2693. It is a valuable clue for cloning the candidate genes related to prostate cancer; Arrhythmogenic Right Ventricular Dysplasia (ARVD); Chediak-Higashi, NK cell Deficiency (CHS); and Hypoparathyrodism with Short Stature, Mental Retardation and Seizures which have already been mapped in this chromosomal region.

  18. Buffalo embryos produced by handmade cloning from oocytes selected using brilliant cresyl blue staining have better developmental competence and quality and are closer to embryos produced by in vitro fertilization in terms of their epigenetic status and gene expression pattern.

    Science.gov (United States)

    Mohapatra, Sushil K; Sandhu, Anjit; Neerukattu, Venkata S; Singh, Karn P; Selokar, Naresh L; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

    2015-04-01

    We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB-). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB- oocytes (43.41 ± 2.54 vs. 22.74 ± 1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB- blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB- blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB- blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB- blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

  19. Embryo-maternal communication

    DEFF Research Database (Denmark)

    Østrup, Esben; Hyttel, Poul; Østrup, Olga

    2011-01-01

    Communication during early pregnancy is essential for successful reproduction. In this review we address the beginning of the communication between mother and developing embryo; including morphological and transcriptional changes in the endometrium as well as epigenetic regulation mechanisms...... directing the placentation. An increasing knowledge of the embryo-maternal communication might not only help to improve the fertility of our farm animals but also our understanding of human health and reproduction....

  20. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  1. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

  2. Cloning and characterization of the human integrin β6 gene promoter.

    Directory of Open Access Journals (Sweden)

    Mingyan Xu

    Full Text Available The integrin β6 (ITGB6 gene, which encodes the limiting subunit of the integrin αvβ6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5'-RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region -24 to -18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located -289 to -150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis.

  3. Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

    Science.gov (United States)

    Bonthron, D T; Brady, N; Donaldson, I A; Steinmann, B

    1994-09-01

    Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

  4. Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: A randomized controlled trial

    NARCIS (Netherlands)

    E.B. Baart (Esther); E. Martini (Elena); M.J.C. Eijkemans (René); D. van Opstal (Diane); N.G.M. Beckers (Nicole); A. Verhoeff (Arie); N.S. Macklon (Nick); B.C.J.M. Fauser (Bart)

    2007-01-01

    textabstractBACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled

  5. The cloning and preliminarily functional analysis of the human neurotrimin gene

    Institute of Scientific and Technical Information of China (English)

    LIU; Jin; LI; Guangtao; PENG; Xiaozhong; LIU; Bingyan; YIN

    2004-01-01

    Proteins of the immunoglobulin superfamily (IgSF) are involved in a variety of specific cell-cell interactions in the developing nervous system. To identify and characterize new members of this protein family in human nervous system, we screen the human fetal brain cDNA library and isolate a full-length cDNA clone which contains a 1032 bp open reading frame encoding a protein of 344 amino acids. Sequence analysis reveals that it is a glycoprotein comprised of three C2-like immunoglobulin domains and is anchored to the plasma membrane via a post-translationally attached glycosyl-phosphatidylinositol (GPI) moiety. The protein shows high sequence similarity to the rat Ntm (97%), so we term it human neurotrimin (NTM). Northern blot analysis reveals that (HUMAN)NTM has three different transcripts with the length of 3.2 kb, 4.0 kb and 9.0 kb respectively. It has a wider expression pattern than that of (RAT) Ntm. Notably, the expression of NTM in fetal brain is higher than that in mature brain and is stronger in nervous tumors than that in normal brain tissues. We insert an HA epitope tag between the third Ig-like domain of NTM and the site of GPI attachment, then construct it into the eukaryotic expression vector pcDNA3.1+/Zeocin. The pcDNA3.1-HA-NTM is transfected into the Chinese Hamster Ovary (CHO) cells. The results demonstrate that HA-NTM is expressed on the surface of CHO cells and could strengthen the aggregation of CHO-NTM cells.

  6. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  7. Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs.

    Science.gov (United States)

    Chumakov, I M; Zabarovsky, E R; Prassolov, V S; Mett, V L; Kisselev, L L

    1982-01-01

    A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage lambda Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.

  8. Cloning and expression of retinoblastoma-binding protein 4 gene in embryo diapause termination and in response to salinity stress from brine shrimp Artemia sinica.

    Science.gov (United States)

    Wang, Xiaolu; Yao, Feng; Liang, Xiaoyu; Zhu, Xiaolin; Zheng, Ren; Jia, Baolin; Hou, Lin; Zou, Xiangyang

    2016-10-15

    Retinoblastoma binding protein 4 (RBBP4) is a nuclear protein with four WD-repeat sequences and thus belongs to a highly conserved subfamily of proteins with such domains. This retinoblastoma-binding protein plays an important role in nucleosome assembly and histone modification, which influences gene transcription and regulates cell cycle and proliferation. Artemia sinica (brine shrimp) undergoes an unusual diapause process under stress conditions of high salinity and low temperature. However, the role of RBBP4 in diapause termination of embryo development in A. sinica remains unknown. Here, the full-length cDNA of the As-rbbp4 gene was obtained from A. sinica and found to contain 1411 nucleotides, including a 1281 bp open reading frame (ORF), 63 bp 5'-untranslated region (UTR) and a 67-bp 3'-UTR, which encodes a 427 amino acid (48 kDa) protein. Bioinformatic analysis indicated As-RBBP4 to be mainly located in the nucleus, with a theoretical isoelectric point of 4.79. Protein sequence domain analysis showed that As-RBBP4 is a conserved protein, especially in the WD40 domain. No specificity in expression of this gene was observed in tissues or organs by in situ hybridization. Real-time quantitative PCR and Western blot analyses of As-RBBP4 gene and protein expression, respectively, showed notably high levels at 10 h and a subsequent downward trend. Obvious trends in upregulation of As-RBBP4 were observed under conditions of low temperature and high salinity stress. As-E2F1 and As-CyclinE also presented similar trends as that of As-RBBP4 in Western blots. Analysis of the RBBP4 expression in early embryonic development of A. sinica indicated that this protein plays an important role in diapause termination and cell cycle regulation.

  9. Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

    DEFF Research Database (Denmark)

    Tao, Yong; Gheng, Lizi; Zhang, Meiling

    2008-01-01

    The low efficiency of somatic cell nuclear transfer may be related to the ultrastructural deviations of reconstructed embryos. The present study investigated ultrastructural differences between in vivo-produced and cloned goat embryos, including intra- and interspecies embryos. Goat ear fibroblast...

  10. MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro.

    Science.gov (United States)

    Feng, Ruizhi; Sang, Qing; Zhu, Yan; Fu, Wei; Liu, Miao; Xu, Yan; Shi, Huijuan; Xu, Yao; Qu, Ronggui; Chai, Renjie; Shao, Ruijin; Jin, Li; He, Lin; Sun, Xiaoxi; Wang, Lei

    2015-03-03

    Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.

  11. FISH analysis of 15 chromosomes in human day 4 and 5 preimplantation embryos : the added value of extended aneuploidy detection

    NARCIS (Netherlands)

    Baart, E. B.; van den Berg, I.; Martini, E.; Eussen, H. J.; Fauser, B. C. J. M.; Van Opstal, D.

    2007-01-01

    Objective Screening for an increased number of chromosomes may improve the detection of abnormal embryos and thus contribute to the capability of preimplantation genetic screening (PGS) to detect the embryo(s) for transfer in IVF with the best chance for a healthy child. Good-quality day 4 and 5 emb

  12. Why Clone?

    Science.gov (United States)

    ... How might cloning be used in medicine? Cloning animal models of disease Much of what researchers learn ... issue of the genetic reshuffling that happensduring sexual reproduction and simply clone our drug-producing cow. Cloning ...

  13. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  14. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    Science.gov (United States)

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  15. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    GU Fu; LI YuYang; L(U) Hong; YOU Chun; LIU JianPing; CHEN Ao; YU Yao; WANG Xiang; WAN DaFang; GU JianRen; YUAN HanYing

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E.coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope α-32p-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  16. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recom-bination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepato-cellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepato-carcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-32P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  17. Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30.

    Science.gov (United States)

    Wang, Jianye; Huang, Yu; Zhou, Mingxu; Hardwidge, Philip R; Zhu, Guoqiang

    2016-06-21

    Muscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood. The FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D' sequence at the 3' ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains

  18. Enhance beef cattle improvement by embryo biotechnologies.

    Science.gov (United States)

    Wu, B; Zan, L

    2012-10-01

    Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

  19. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  20. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

    Science.gov (United States)

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

    2016-01-01

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  1. The impact of commercialisation on public perceptions of stem cell research: exploring differences across the use of induced pluripotent cells, human and animal embryos.

    Science.gov (United States)

    Critchley, Christine R; Bruce, Gordana; Farrugia, Matthew

    2013-10-01

    The development of pluripotent cells that enable stem cell research (SCR) without destroying human embryos is now a leading priority for science. Public and political controversies associated with human embryonic SCR experienced in the recent past should be alleviated if scientists no longer need to harvest cells from human embryos. This research suggests however additional issues needing attention in order to gain the public's trust and support: the use of mouse embryos and the commercialisation of research. Using a representative sample of 2,800 Australians, and an experimental telephone survey design, this research compared levels and predictors of public support for stem cell research across three cell source conditions: human embryo (HE), mouse embryo (ME) and induced pluripotent cells (iPSCs). The results revealed that the public were significantly more likely to support research using iPSCs than HE and ME cells and public compared to private research (regardless of the cell source). There was no significant difference in support for HE compared to ME research, but the former was viewed as more likely to lead to accessible health care benefits and to be associated with more trustworthy scientists. The results of a multimediation structural equation model showed that the primary reason support for SCR significantly dropped in a private compared to public context (i.e., the commercialisation effect) was because public scientists were trusted more than private scientists. This effect was consistent across all three SCR materials, suggesting that the use of mouse embryos or even iPSCs will not reduce the publics' concern with commercialised science. The implications these results have for public acceptance of stem cell and animal research are discussed in relation to possible solutions such as increasing public awareness of the regulation of animal research and benefit sharing.

  2. Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

    Science.gov (United States)

    Shrestha, Pravesh; Yun, Ji-Hye; Kim, Woo Taek; Kim, Tae-Yoon; Lee, Weontae

    2016-03-01

    A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

  3. Cloning,sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

    Institute of Scientific and Technical Information of China (English)

    ZHANGJINSONG; MINGYEH

    1994-01-01

    The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.

  4. The cloning, genomic organization and tissue expression profile of the human DLG5 gene

    Directory of Open Access Journals (Sweden)

    Gibbs Richard A

    2002-02-01

    Full Text Available Abstract Background Familial atrial fibrillation, an autosomal dominant disease, was previously mapped to chromosome 10q22. One of the genes mapped to the 10q22 region is DLG5, a member of the MAGUKs (Membrane Associated Gyanylate Kinase family which mediates intracellular signaling. Only a partial cDNA was available for DLG5. To exclude potential disease inducing mutations, it was necessary to obtain a complete cDNA and genomic sequence of the gene. Methods The Northern Blot analysis performed using 3' UTR of this gene indicated the transcript size to be about 7.2 KB. Using race technique and library screening the entire cDNA was cloned. This gene was evaluated by sequencing the coding region and splice functions in normal and affected family members with familial atrial fibrillation. Furthermore, haploid cell lines from affected patients were generated and analyzed for deletions that may have been missed by PCR. Results We identified two distinct alternately spliced transcripts of this gene. The genomic sequence of the DLG5 gene spanned 79 KB with 32 exons and was shown to have ubiquitous human tissue expression including placenta, heart, skeletal muscle, liver and pancreas. Conclusions The entire cDNA of DLG5 was identified, sequenced and its genomic organization determined.

  5. Cloning and expression of MXR7 gene in human HCC tissue

    Institute of Scientific and Technical Information of China (English)

    Xue Ping Zhou; Hong Yang Wang; Guang Shun Yang; Zheng Jun Chen; Bao An Li; Meng Chao Wu

    2000-01-01

    AIM To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using 32P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively.The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC <5cm was 90% (9/10) and 83.3% (5/6),respectively.CONCLUSION MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.

  6. Improving embryo quality in assisted reproduction

    NARCIS (Netherlands)

    Mantikou, E.

    2013-01-01

    The goal of this thesis was to improve embryo quality in assisted reproductive technologies by gaining more insight into human preimplantation embryo development and by improving in vitro culture conditions. To do so, we investigated an intriguing feature of the human preimplantation embryo, i.e. it

  7. Detection of cancer clones in human gastric adenoma by increased DNA-instability and other biomarkers

    Directory of Open Access Journals (Sweden)

    A Sun

    2009-06-01

    Full Text Available An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30°C for 20 min (DNA-instability test has been used as a marker of malignancy. The test was applied to bioptic tissues of human gastric polyp assessed histopathologically as foveolar hyperplastic polyp (13 cases, mild (58 cases, moderate (86 cases, and severe (20 cases dysplasia, and adenocarcinomas (14 cases. The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor (DFF45, and basic fibroblast growth factor (bFGF. The DNA-instability test was positive in 14 (100% adenocarinoma cases, 20 (100% severe dysplasia cases, 52 (60.5% moderate dysplasia cases, and 12 (20.7% mild dysplasia cases, indicating malignancy. All foveolar hyperplastic polyps were negative to the DNA-instability testing. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%, to moderate (40%, to severe (100% dysplasia, and adenocarcinoma (100%. All other biological markers tested in the present study showed significantly higher values in the adenoma glands, being positive to DNA-instability testing, irrespective of the dysplasia grade, as compared to those in the adenoma glands that were negative to DNA-instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. These results indicate that cancer cell clones are already present at the adenoma stages showing a positive DNA-instability test, enhanced proliferative activity, p53 mutation, induction of DFF45 and bFGF. These factors allow cancer cell proliferation, producing heterogeneous subclones due to DNA-instability, enhancing their survival by escaping apoptosis, and providing abundant nutrients during the early-stage progression of gastric cancer. Based on these findings, we herein propose the

  8. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols.

    Science.gov (United States)

    Mei, Xin; Wu, Yuan-Yuan; Mao, Xiao; Tu, You-Ying

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green te