Sample records for aminopterin

  1. Teratogenicity studies with methotrexate, aminopterin, and acetylsalicylic acid in domestic cats. (United States)

    Khera, K S


    Pregnancy was timed in cats following induced ovulation. Methotrexate, (0.5 mg/kg), aminopterin, (0.1 mg/kg), and acetylsalicylic acid, (25 or 50 mg/kg) were administered orally in gelatin capsules in single daily doses on different days of gestation, methotrexate (MTX) on days 11-14, 14-17, or 17-20, aminopterin on day 12, 14, or 16, and acetylsalicylic acid (ASA) on days 10-15 or 15-20. Maternal toxicity was produced only by MTX. MTX given on days 11-14 and 14-17 produced high frequencies of malformations including umbilical hernia. Aminopterin caused no conclusive teratogenic response. An overall increased frequency of anomalies occurred after 50 mg/kg ASA but no single anomaly predominated.

  2. Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

    International Nuclear Information System (INIS)

    The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated

  3. Tritiated thymidine and deoxycytidine suicide of mouse hemopoietic colony forming cells (CFC)

    International Nuclear Information System (INIS)

    Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1-100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd. (author)

  4. Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry. (United States)

    Nikolic, D; van Breemen, R B


    A method of screening combinatorial libraries for inhibitors of eukaryotic dihydrofolate reductase has been developed using pulsed ultra-filtration electrospray mass spectrometry, which is a continuous-flow affinity separation system for extracting and identifying high affinity ligands in combinatorial libraries. In this application, pulsed ultrafiltration conditions were optimized for the isolation and identification of inhibitors of dihydrofolate reductase from a 22 compound library containing six known inhibitors of the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine, folic acid, and folinic acid, and 16 compounds without known affinity. In order to optimize the screening method, sources of non-specific binding were identified and minimized. A significant source of non-specific binding for this set of library compounds was hydrophobic interaction with the surfaces of the ultrafiltration chamber. After affinity separation of bound (high affinity) versus free (low affinity) library compounds during pulsed ultrafiltration, receptor-bound ligands were released and eluted using either organic solvent or acidified mobile phase. Although 80% methanol easily disrupted the receptor-ligand complexes, organic solvent had the undesirable effect of releasing non-specifically bound compounds from the chamber and thereby increasing the background noise. Interference from non-specific binding was minimized by releasing bound ligands using a low pH mobile phase eluent instead of organic solvent. Under the conditions used, pulsed ultrafiltration mass spectrometry selectively identified the two library compounds with the highest affinity for dihydrofolate reductase, methotrexate and aminopterin.

  5. 'Rod and ring' formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway. (United States)

    Calise, S John; Purich, Daniel L; Nguyen, Thuy; Saleem, Dania A; Krueger, Claire; Yin, Joyce D; Chan, Edward K L


    'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway. PMID:27343244

  6. Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G (United States)

    Larimer, Cassandra M.; Slavnic, Dejan; Pitstick, Lenore D.; Green, Jacalyn M.


    Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in

  7. Biochemical transformation of deoxythymidine kinase-deficient mouse cells with uv-irradiated equine herpesvirus type 1

    Energy Technology Data Exchange (ETDEWEB)

    Allen, G.P.; McGowan, J.J.; Gentry, G.A.; Randall, C.C.


    A line of 3T3 mouse cells lacking deoxythymidine kinase (dTK/sup -/) was stably transformed to the dTK/sup +/ phenotype after exposure to uv-irradiated equine herpesvirus type 1 (EHV-1). Biochemical transformants were isolated in a system selective for the dTK/sup +/ phenotype (Eagle minimal essential medium containing 10/sup -4/ M hypoxanthine, 6 x 10/sup -7/ M aminopterin, and 2 x 10/sup -5/ M deoxythymidine). Transformation was accompanied by the acquisition of a dTK activity with immunological, electrophoretic, and biochemical characteristics identical to those of the dTK induced by EHV-1 during productive infection. The transformed cells have been maintained in selective culture medium for more than 50 passages and have retained the capacity to express EHV-1-specific antigens. Spontaneous release of infectious virus has not been detected in the transformed lines, and the cells were not oncogenic for athymic nude mice. In contrast to normal dTK/sup +/ 3T3 cells, EHV-1 transformants were unable to grow in the presence of arabinosylthymine, a drug selectively phosphorylated by herpesvirus-coded dTK's. These results indicate that a portion of the EHV-1 genome is able to persist in the transformed cells for many generations and be expressed as an enzymatically active viral gene product.

  8. Analogues of methotrexate in rheumatoid arthritis. 1. Effects of 10-deazaaminopterin analogues on type II collagen-induced arthritis in mice. (United States)

    DeGraw, J I; Colwell, W T; Crase, J; Smith, R L; Piper, J R; Waud, W R; Sirotnak, F M


    Carbonation of the dianions (LDA) of 5-methylthiophene-2-carboxylic, 2-methylpyridine-5-carboxylic, and 3-methylpyridine-6-carboxylic acids provided the respective carboxy heteroarylacetic acids. The crude diacids were directly esterified in MeOH-HCl to afford the diesters. Alkylation of the sodio anions with ethyl iodide yielded the appropriate alpha-ethyl diesters. The anions of the various diester substrates were then alkylated by 2,4-diamino-6-(bromomethyl)-pteridine followed by ester saponification at room temperature to afford the respective 2,4-diamino-4-deoxy-10-carboxy-10-deazapteroic acids. The 10-carboxyl group was readily decarboxylated by heating in DMSO at temperatures of 110-135 degrees C to give the diamino 10-deaza heteropteroic acid intermediates. Coupling with diethyl L-glutamate followed by ester hydrolysis afforded the target aminopterins. The analogues were evaluated for antiinflammatory effect in the mouse type II collagen model. The thiophene analogue of 10-ethyl-10-deazaaminopterin was found to be an effective inhibitor in terms of reduced visual evidence of inflammation and swelling as determined by caliper measurement. PMID:9022804

  9. Preparation and Preliminary Biological Evaluation of {sup 177}Lu-DOTA folate as Potential Folate Receptor Targeting Therapeutic Agent

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    Choi, Kang-Hyuk; Hong, Young-Don; Pyun, Mi-Sun; Lee, So-Young; Felipe, Fenelope; Yoon, Sun-Ha; Choi, Sun-Ju [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)


    Folic Acid (FA) and FA derivatives are overexpressed on several tumor cells. The cell-membrane folic acid receptors are known to be responsible for the cellular accumulation of FA and FA analogs, such as methotrexate and folic acid. Folate has been characterized to have high affinity for the folate-receptor positive cells and tissues and considered to be useful as diagnostic imaging and therapeutic agent. In 1940s, Folate analogue, aminopterin, was first used for treatment of leukemia and recently, many folate derivatives were tried for cancer-treatment agent as well as visualization of folate receptor. Many researchers tried to conjugate folic acid with macromolecules or low molecular weight chelators through its alpha or gamma carboxylate. However, despite the reduced binding affinity, FAs are still recognized by the folate receptor. Therefore, we focused to develop folate-based radiopharmaceutical that has the potential to be used as a therapeutic agent. We report here the synthesis and the radiolabeling of {sup 177}Lu-DOTA as well as the biodistribution data of our developed compound.

  10. Production of immunoglobin G human monoclonal antibodies by cellular fusion method

    International Nuclear Information System (INIS)

    The increasing clinical use of immunological detection i.e. radioimmunoassays (RIA) enzyme linked immunosorment assay (ELISA) and immunoscintigraphy, has focused the attention on the important of monoclonal antibodies production. Monoclonal antibody (MAB) has been prepared by the cellular fusion method using immunoglobin G human (IgGh) as the antigen and polyethylene glycol (PEG) as the fusion agent. Selection of hybridoma cells was carried out using hypoxanthine-aminopterin-thymidine (HAT) medium. The immunoreactivity and specificy of the antibodies were determined by ELISA technique using Goat anti Mouse (GAM) polyclonal antibodies conjugated with horse-radish peroxidase. It has been shown that the MAB secreted from hybridoma cells are specific to IgGh, IgGh-, IgGh-k, IgG1h, and IgG2h. It was also observed that the MAB are specific to goat IgG or rabbit IgG. All MAB in the ascities fluid were IgG1-k class as determined by ouchterlony method. (author). 6 refs.; 4 tabs.; 5 figs

  11. Effects of sulfanilamide and methotrexate on 13C fluxes through the glycine decarboxylase/serine hydroxymethyltransferase enzyme system in Arabidopsis

    International Nuclear Information System (INIS)

    In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [alpha-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis

  12. Genetic stability of murine pluripotent and somatic hybrid cells may be affected by conditions of their cultivation. (United States)

    Ivanovna, Shramova Elena; Alekseevich, Larionov Oleg; Mikhailovich, Khodarovich Yurii; Vladimirovna, Zatsepina Olga


    Using mouse pluripotent teratocarcinoma PCC4azal cells and proliferating spleen lymphocytes we obtained a new type of hybrids, in which marker lymphocyte genes were suppressed, but expression the Oct-4 gene was not effected; the hybrid cells were able to differentiate to cardiomyocytes. In order to specify the environmental factors which may affect the genetic stability and other hybrid properties, we analyzed the total chromosome number and differentiation potencies of hybrids respectively to conditions of their cultivation. Particular attention was paid to the number and transcription activity of chromosomal nucleolus organizing regions (NORs), which harbor the most actively transcribed - ribosomal - genes. The results showed that the hybrids obtained are characterized by a relatively stable chromosome number which diminished less than in 5% during 27 passages. However, a long-term cultivation of hybrid cells in non-selective conditions resulted in preferential elimination of some NO- chromosomes, whereas the number of active NORs per cell was increased due to activation of latent NORs. On the contrary, in selective conditions, i.e. in the presence of hypoxantine, aminopterin and thymidine, the total number of NOR-bearing chromosomes was not changed, but a partial inactivation of remaining NORs was observed. The higher number of active NORs directly correlated with the capability of hybrid cells for differentiation to cardiomyocytes.

  13. An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

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    Fanning, Sean W.; Horn, James R. (NIU)


    Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

  14. Co-purification of dihydrofolate synthetase and N10formyltetrahydropteroyldiglutamate synthetase from E. coli

    International Nuclear Information System (INIS)

    The enzymatic activities which add a single glutamate to dihydropteroate (H2Pte) and to N10formyltetrahydropteroylglutamate (N10formylH4PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N10formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H2Pte was a noncompetitive inhibitor (Kii . 1.1 microM) of the utilization of N10formylH4PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis . 370 microM) of the reaction with N10formylH4PteGlu but was not inhibitory with H2Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 microM respectively. With N10formylH4PteGlu as substrate, these Km values were 1.2 mM and 80 microM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein

  15. Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

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    L.C. Kreutz


    Full Text Available Three Brazilian isolates of bovine viral diarrhea virus (BVDV, antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs. Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11, were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid and 1:100 (hybridoma culture supernatant in IFA and immunoperoxidase (IPX staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

  16. Sandwich enzyme-linked immunosorbent assay for naringin. (United States)

    Qu, Huihua; Wang, Xueqian; Qu, Baoping; Kong, Hui; Zhang, Yue; Shan, Wenchao; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan


    Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA. PMID:26709308

  17. p53 ser249突变对小鼠EF细胞周期和凋亡的影响%Effect of p53 249 Arg to Ser mutation on cell cycle and apoptosis in mouse EF cells

    Institute of Scientific and Technical Information of China (English)

    吴智群; 李庆霞; 李臻; 于翠娟; 罗亚宁


    Objective To explore the effect of p53 249 Arg to Ser mutation on function of p53 in mouse EF cells.Methods Arg to Ser mutation was introduced into the p53 249 code by knock-in method.The ES cells with p53 249 Arg to Ser mutation were selected according to the homologues-recombination with RCR and Southern blot.The positive ES cells without a selection marker were injected into blastocysts recovered from Hprt-/-mice,which were derived from Hprt-deficient ES cells.The injected blastocysts then were implanted into pseudopregnant females.At embryonic day 14,MEFs were recovered from the embryos and cultured under the selection of HAT (0.016 mg of hypoxanthine/ml,0.01 mM aminopterin,0.0048 mg of thymidine/ml) and the cell cycle,apoptosis,and the relative protein expression were analyzed by flow cytometry and Western blot.Results 1) The EF cells with 249 Arg to Ser mutation were more resistant to the UV induced cell cycle arrest compared to the wild type EF cell (P<0.05).2) The EF cells with 249 Arg to Ser mutation were more resistant to the UV induced apoptosis compared to the wild type ES cell (P<0.05).3) The mutation did not affect the expression of p53 after UV but decrease the expression of BAX and p21 after UV.Conclusion Thep53 249 Arg to Ser mutation can disable the function of p53 but do not change the expression of p53 in EF cells.%目的 研究p53 249编码子突变对小鼠EF细胞p53功能和信号传导的影响.方法 利用基因打靶技术在小鼠ES细胞p53基因249编码子中引入点突变.使编码子249由Arg变成Ser,根据同源重组规律采用PCR或Southern方法筛选带有p53 249突变的阳性ES细胞,并通过测序确定该ES细胞的p53 249编码子已经由Arg变成Ser,然后将含突变而不含筛选标记的ES细胞微注射到从Hprt-/-小鼠收集的囊胚中,将注射过的囊胚植入假孕的雌性小鼠子宫,到第14天取小鼠胚胎EF细胞,用含HAT的培养液[DMEM含10%FCS,glutamine,antibiotics,50 mM mecaptoethanol