摘要
以F4代转hGH基因红鲤体细胞 (肾脏和尾鳍 )及培养 18代的F4代转hGH基因红鲤尾鳍细胞为核供体 ,泥鳅或黄河鲤成熟卵为受体 ,进行了核移植 ,以探讨外源F4代转基因鱼体外源基因的分布与存在形式、稳定性和克隆转基因鱼的可能性。F4代红鲤肾脏细胞核与泥鳅卵配合的核移植胚胎有 12 4 %发育到囊胚 ,0 33%发育到神经胚 ;F4代尾鳍细胞核移入泥鳅卵后的重组胚发育到囊胚、神经胚、肌节期和肌肉效应期的胚胎分别为 2 4 5 %、0 3%、0 2 %和 0 1% ;对照卵无发育。F4代红鲤尾鳍培养细胞与黄河鲤卵子配合的重组胚胎有 5 0 5 3%发育到囊胚 ,5 6 9%发育到原肠胚 ,0 5 3%发育到神经胚 ,0 4 %发育到肌节期。说明由于同种细胞核与卵细胞的相容性高于异种核卵的相容性 ,早期发育率高 ;而由于培养细胞的异倍化 ,后期的发育率降低。用PCR技术对供体鱼不同个体及同一个体不同组织外源基因检测 ,结果 10 0 %个体为阳性鱼 ,而且不同组织的阳性率也是 10 0 % ,说明外源基因均匀分布在不同组织中。无论F4代转基因鱼的肾脏细胞、尾鳍细胞还是培养的尾鳍细胞作核移植供体 ,核移植胚胎中hGH基因的检出率为 10 0 %。说明F4代转基因红鲤个体不同细胞都存在hGH基因 ,而且经长期培养不会丢失。表明F4代转基因红鲤中的外源hGH基?
Nuclear transplantation was performed using kidney cells, tail fin cells and cultured tail fin cells of 18th passage of the F 4hGH gene transferred red carp (\$Cyprinus carpio haematopterus\$) as donors and the eggs of loach (\$Misgurnus anguillicaudatus\$) of Huanghe carp (\$Cyprinus carpio haematopterus\$) as recipients. Using loach unfertilized eggs as recipients 0\^33% of reconstructed embryos developed in to neural plate from kidney cells, and 0\^1% of reconstructed embryos developed in to muscular reaction stage from tail fin cells of F 4 hGH gene transferred red carp. A tatal of 0\^4% reconstructed embryos from cultured tail fin cells of 18th passage and unfertilized eggs of Huanghe carp developed in to myotome stage. All the nuclear transfer embryos were positive for hGH gene by PCR analysis. The results suggest that exogenous hGH gene is stable in the F 4 hGH gene transferred red carp, hGH gene exists in all the cells of the F 4 transgenic red carp. Somatic cell nuclear transfer is an effective pathway for producing homogenous transgenic fish, but with low efficiency. The reasons of low efficiency of somatic nuclear transplantation may be incompatibility of nuclear and ooplasm, incongruity of cell cycle, aberrance of chromosomes, etc.
基金
国家自然科学基金重点项目 (3 973 0 2 90 )资助 ~~
关键词
转基因红鲤
体细胞
核移植
鲤鱼
泥鳅
同质化
fish
carp
loach
somatic cell
nuclear transfer, transgene
homogenous
