title: [Production of transgenic embryos through nuclear transfer using ovine fetal fibroblasts transferred with foreign genes].
abstract: Human ALR gene sequence was amplified by PCR from human total DNA and inserted into pIRES(2)-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo(r) and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/ALR by the induction of lipofectAMINE(TM). The positive cell clones were selected with medium containing G418 (800 μg/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and immuno-histochemical staining. Results showed that the EGFP and ALR genes linked with IRES were coexpressed simultaneously in sFFCs; the blastocysts formed by nuclear transfer using tranfected donor cells are all transgenic blastocysts. EGFP, ALR and Neo(r) gene were all expressed in the transgenic embryos. In conclusion that a method to construct the positive embryos before pre-implantation which stably express ALR gene by the indication of EGFP expression has been successfully established. The application of this method can simplify the procedure of testing the targets and contribute to the efficiency increasing of transgenic domestic animal production.
authors.affiliation: Inner Mongolia Hospital, Hohhot, 010017, China. mayz@imnu.edu.cn
authors.forename: Yu-Zhen
authors.initials: YZ
authors.lastname: Ma
authors.forename: Yu
authors.initials: Y
authors.lastname: Ren
authors.forename: Xue-Yuan
authors.initials: XY
authors.lastname: Zhou
authors.forename: Dong-Jun
authors.initials: DJ
authors.lastname: Liu
authors.forename: Ri-Gan
authors.initials: RG
authors.lastname: Xu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012756:Sheep / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D050862:Oxidoreductases Acting on Sulfur Group Donors
chemical_list: D049452:Green Fluorescent Proteins; D011506:Proteins; C088825:GFER protein, rat; D050862:Oxidoreductases Acting on Sulfur Group Donors