Basic and applied research on production of "double-muscle" cattle by use of a chimeric DNA/RNA oligo-nucleotide

下面进行了利用DNA/RNA嵌合寡核苷酸生产转化生长因子8基因（TGF8）敲除牛的研究。1)为了研究受体细胞、牛成纤维细胞和积云细胞的细胞周期对基因表达的影响，在细胞核内注射p-β-act/luc^+/IRES/EGFP/neo^r。当细胞处于融合状态时，荧光被高度检测到，而血清饥饿后则没有。为了检验细胞的基因转化效率，采用基因导入方法、dna显微注射、电泳和转染试剂将p-β-act/luc^+/IRES/EGFP/neo^r基因导入牛体细胞。用转染试剂检测EGFP荧光，转染效果最好。此外，在转染的细胞中，G418选择后进一步检测荧光，并在转染的核移植胚胎中检测荧光。这表明该基因可能更多地整合在细胞基因组中。从数据上看，使用转染试剂将外源基因导入牛体细胞是最有效的。2)酵母编码核糖体蛋白L29的cyh2点突变诱导了对环氯己亚胺的耐受性。我们检测了哺乳动物核糖体蛋白L27的同源基因的类似突变是否在细胞培养过程中诱导了对环氯己亚胺的耐受。设计了诱导L27基因突变的嵌合DNA/RNA寡核苷酸，用转染试剂将其导入牛和小鼠成纤维细胞，在环氯己亚胺环境下培养10 d。未转染和载体转染的细胞在环氯肟胺培养10天后均发生变性，但转染寡核苷酸的牛和小鼠细胞即使在环氯肟胺培养10天后也能存活。结果表明，转染该寡核苷酸可诱导细胞L27基因发生点突变，使细胞获得对环氯己亚胺的耐受性。3)设计了诱导牛GDF8基因点突变的嵌合DNA/RNA寡核苷酸。寡核苷酸的使用可以提供生产KO动物的替代方法。少

The following research was done for the production of transforming growth factor 8 gene (TGF8) knock-out cattle using a chimeric DNA/RNA oligo-nucleotide.1) To investigate effects of cell cycle of the recipient cells, bovine fibroblasts and cumulus cells, on the gene expression, p-β-act/luc^+/IRES/EGFP/neo^r was injected into the cell nuclei. The luminescence was highly detected when cells were in a confluent state, but not after serum starvation. To examine the efficiency of gene transformation of the cells, gene introduction methods, DNA-microinjection, etectroporation and a transfection reagent were used for introduction of the p-β-act/luc^+/IRES/EGFP/neo^r into the bovine somatic cells. The gene was transfected most effectively by use of the transfection reagent by detecting EGFP fluorescence. Furthermore, in the transfected cells, the fluorescence was further detected after selection by G418, and in nuclear transplanted-embryos with the transfected. These indicated that the gene m … More ay be integrared in genome of the cells. From the data, the use of the transfection reagent was the most effective to introduce the exogenous gene into bovine somatic cells.2) A point mutation of cyh2 encoding L29 which is a ribosomal protein in yeast induce cychloheximide tolerance. We examined whether the similar mutation of the homologous gene encoding L27, a mammalian ribosomal protein induced the cychloheximide tolerance during cell culture. We designed the chimeric DNA/RNA oligo-nucleotide which induced the mutation of L27 gene, and introduced the oligo-nucleotide into bovine and mouse fibroblasts by the transfection reagent The cells were cultured under cychloheximide for 10 days. All of the not-transfected and vecter-transfected cells were degenerated 10 days after culture with cychloxeximide, but bovine and mouse cells transfected with the oligo-nucleotide were survived even with cychloheximide for 10 days. The results showed the transfection of the oligo-nucleotide might induce the point-mutation of the L27 gene of the cells and consequently the cells acquired the cychloheximide tolerance.3) We designed the the chimeric DNA/RNA oligo-nucleotide which induced point-mutation of bovine GDF8 gene. The use of the oligo-nucleotide may provide the alternative method to produce the KO animals. Less