SPARC基因敲除小鼠的繁育及子代基因型的鉴定  

Breeding of SPARC Knockout Mice and Gene Identification of Filial Generation

作　　者：罗盘[1] 罗振[1] 赵秀华[1] 肖娜[1] 程腊梅[1,2] 

机构地区：[1]中南大学生殖与干细胞工程研究所,湖南长沙410078 [2]人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《现代生物医学进展》2010年第3期408-410,共3页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30771053);中南大学研究生创新基金(2009ssxt103)

摘　　要：目的:探讨富含半胱氨酸的分泌型酸性蛋白(SPARC)基因敲除小鼠的繁殖和鉴定方法,为进一步研究SPARC蛋白的功能奠定基础。方法:将所引进的杂合子小鼠进行饲养并繁殖,繁殖成功后其子代中将会出现野生型、杂合子以及纯合子3种基因型。提取子鼠鼠尾基因组DNA,用PCR法扩增SPARC和Neo基因片段,通过限制性内切酶酶切反应,证实PCR扩增产物的正确性;应用Westernblot检测SPARC蛋白质的表达,进一步证实PCR鉴定方法的可靠性。结果:SPARC基因杂合子小鼠互交繁殖结果基本符合孟德尔遗传规律,SPARC基因缺失纯合子小鼠有繁殖能力。琼脂糖凝胶电泳结果显示PCR产物分子量大小与预期的目的基因片段相对分子质量大小一致,酶切和Westernblot结果证实PCR结果可靠。结论:PCR方法能够准确鉴定SPARC基因突变小鼠基因型。Objective：To explore the ideal breeding way of Secreted Protein Acidic and Rich in Cysteine （SPARC） knockout mice and identify mice with SPARC gene mutant for laying a foundation for studying the role of SPARC. Methods：Breed and propagate the introduced heterozygote mice. Crosses between heterozygotes produced three genotype offsprings： heterozygotes, homozygous and wild-type. The genome DNA extracted from the tails of filial generation mice and PCR method was employed to amplify the SPARC and Neo gene fragments. Then, we analyzed the specificity of PCR primers through digesting PCR products with restriction endonuclease. Additionally, the expression of SPARC protein was detected by Western blotting to further confirmed the reliability of PCR methods for genotype identification of SPARC mutant mice. Results：Crosses between heterozygotes produced a Mendelian distribution of wild-type （23.5%）, heterozygous （50%）, and homozygous（26.4%） mutants. The homozygote mice were fertile. Electrophoresis results revealed that the molecular weight of PCR product was in accordance with the expected gene fragment. The results of restriction endonuclease enzyme digestion and Western blotting confirmed the reliability of PCR methods for genotype identification of SPARC knock out mice. Conclusion：PCR methods can identify the genotype of the mice with SPARC gene mutant precisely .

关 键 词：富含半胱氨酸的分泌型酸性蛋白 基因敲除 基因型鉴定 

分 类 号：Q95-3[生物学—动物学] Q75