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

利用嵌合DNA/RNA寡核苷酸生产“双肌”牛的基础与应用研究

基本信息

批准号：
12660261
负责人：
SAEKI Kazuhiro
金额：
$ 2.24万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660261/
关键词：
TGP8 Cattle embryo clone transfection somatic cells

项目摘要

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

本研究采用嵌合DNA/RNA寡核苷酸构建转化生长因子8（TGF 8）基因敲除牛模型，研究了细胞周期对TGF 8基因表达的影响。1）将p-β-act/luc^+/IRES/EGFP/neo^r基因注入受体牛成纤维细胞和卵丘细胞核内。当细胞处于汇合状态时，高度检测到发光，但血清饥饿后没有。为了检测细胞的基因转化效率，采用基因导入方法、DNA显微注射、电穿孔和转染试剂将p-β-act/luc^+/IRES/EGFP/neo^r导入牛体细胞。通过检测EGFP荧光，使用转染试剂最有效地转染基因。转染细胞经G418筛选后，进一步检测到荧光，转染细胞的核移植胚中也检测到荧光。这些结果表明， ...更多信息可以整合到细胞的基因组中。从数据来看，使用转染试剂将外源基因导入牛体细胞是最有效的。2）编码酵母核糖体蛋白L29的cyh 2的点突变可诱导环己酰亚胺耐受性。我们研究了在细胞培养过程中，编码哺乳动物核糖体蛋白L27的同源基因的类似突变是否诱导了环磷酰胺耐受性。我们设计了一种能诱导L27基因突变的嵌合DNA/RNA寡核苷酸，并通过转染试剂将其导入牛和小鼠成纤维细胞中。所有未转染和载体转染的细胞在用环磷酰胺培养10天后退化，但用寡核苷酸转染的牛和小鼠细胞即使用环磷酰胺培养10天也存活。结果表明，该寡核苷酸的转染可诱导细胞L27基因点突变，从而获得环磷酰胺耐受性。3）设计了诱导牛GDF 8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产生KO动物的替代方法。少