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KMID : 0900920000240040429
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Korean journal of Animal Reproduction
2000 Volume.24 No. 4 p.429 ~ p.437
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Production of Transgenic Bovine Embryos Following Nuclear Transfer of Bovine Fetal Fibroblasts Transfected by Foreign Genes
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Kil K.-S.
Uhm S.-J.
Kim E.-H.
Chung H.-J.
Kim T.-H.
Park H.-D.
Lee H.-T.
Chung K.-S.
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Abstract
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This study investigated the successful introduction of genes of erythropoietin (EPO) and enhanced green fluorescent protein (EGFP) in bovine embryos following nuclear transfer of bovine fetal fibroblasts (bFF), which were transfected by retrovirus vector system. Non-starved bFF were, transferred into perivitelline space of enucleated oocytes. The bFF-oocyte units were accomplished by cell to cell fusion and activated with calcium inophore and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CRlaa medium for 8 days. Out of 187 (EPO) and 210 (EGFP) bovine eggs reconstructed by nuclear transfer, 149 (EPO : 80.0%) and 158 (EGFP : 75.2%) embryos were cleaved, and among them 36 (EPO : 24.2%) and 35 (EGFP : 22.2%) embryos developed to the blastocyst stage. Of these blastocysts, 100% integration of EPO gene in 36 embryos was determined by PCR, and 100% expression of EGFP gene in 35 embryos was observed under the fluorescent microscope. This result indicates that bovine oocytes reconstructed by nuclear transfer of transfected bFF can successfully develop to the blastocyst stage. Furthermore, this novel procedure may be presumably an attractive method efficiently to produce the transgenic cattles.
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KEYWORD
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Nuclear transfer, Fetal fibroblast, EPG, EGFP, Bovine
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