2 mol/L尿素溶血试验在UT-B基因敲除小鼠核型鉴定中的应用

目的 探讨2mol/L尿素溶血试验在UT-B基因敲除小鼠核型鉴定中的作用.方法 (1)按SP级动物饲养标准进行饲养和繁殖;以UT-B基因敲除纯合子(UT-B-/-)成年雄鼠与C57/bl雌鼠合笼,繁殖出UT-B基因敲除杂合型小鼠(UT-B /-,F1代),F1代UT-B /-雄、雌鼠交配获F2代小鼠.(2)取F2代小鼠剪尾提取基因组DNA进行核型鉴定筛选纯合子小鼠(UT-B-/-)和野生型小鼠(UT-B / );(3)应用2 mol/L尿素溶血试验和肾脏RT-PCR进行UT-B-/-(Jknull)表型鉴定.结果 (1)在SP级饲养和繁殖的条件下,已获得足够的UT-B-/-和UT-B / 小鼠;(2)2 mol/L尿素溶血试验证明,UT-B / 小鼠和UT-B /-小鼠的红细胞加入到2mol/L尿素中立刻溶血,UT-B-/-小鼠的红细胞10 min内不溶显示溶血抵抗.结论 2 mol/L尿素溶血试验可准确地鉴定出UT-B-/-小鼠,与基因组DNA的PCR鉴定方法联合应用,可以使引进的UT-B-/-小鼠稳定繁殖、传代.

2 mol/L urea solution hemolysis test in the genotype identification of UT-B knockout mice

Objective To investigate the contribution of 2 mol/L urea solution hemolysis test in the genotype identification of UT-B knockout mice.Methods UT-B knockout heterozygote mice(F1) were bred and Genomic DNA was extracted from the murine tails of F1's offsprings(F2).The Genomic DNA PCR was carried out for genotype identification.The phynotype of F2 generation was identified by 2 mol/L urea solution hemolysis test and RT-PCR.Results UT-B knockout mice were successful bred according to specific pathogen free animal(SP) standard.After genotype identification,more UT-B knockout heterozygote mice were selected.Red blood cells of UT-B knockout mice appeared haemolytic resistivity to 2 mol/L urea solution while wild type mice and heterozygote mice were haemolytic immediately in that solution.No UT-B mRNA was detected from UT-B knockout mice's kidney.Conclusion 2 mol/L urea solution hemolysis test was an appropriate methods for identify UT-B knockout mice,associate with genomic DNA PCR results in the breeding and identification of UT-B knockout mice stable and exact.