【摘 要】
：
根据已知大鼠次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hypoxanthine Guanine Phosphoribosyl Transferase,HPRT)基因的外显子序列,从大鼠HPRT基因组DNA序列的细菌人工染色体(Bacterial
【机 构】
：
内蒙古大学生命科学学院,Monash Institute of Reproduction and Development
论文部分内容阅读
根据已知大鼠次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hypoxanthine Guanine Phosphoribosyl Transferase,HPRT)基因的外显子序列,从大鼠HPRT基因组DNA序列的细菌人工染色体(Bacterial Artificial Chromosome,BAC)中用酶切和PCR方法分别分离得到用于构建基因敲除载体的3.0 kb的5′长臂(Long Arm,LA)和1.7 kb的3′短臂(Short Arm,SA),并分别克隆到pSL1180和pCR2.1中.进一步构建大鼠HPRT基因打靶载体--pKO-HPRT,经酶切鉴定后的大鼠HPRT基因敲除载体用NotⅠ酶切使其线性化,经溴乙锭、正丁醇、酚、酚/氯仿提纯后,将终浓度调至1 μg/μl.在FuGene-6转染试剂的作用下转染培养24 h的第二代大鼠胎脑神经干细胞(Rat Fetal Neural Stem Cells,rFNSCs).转染后的细胞用80 μg/ml G418和0.2 μmol/L的Ganc全培养液筛选,2 w后将存活细胞进行悬浮培养,使细胞形成球形物,挑选单个的球形物进行单克隆增殖,其中一部分细胞(约2～3×103)用裂解液处理,取上清用于PCR检测,大部分细胞(5×107)用于DNA和RNA的提取,进行Southern bolt和RT-PCR检测,剩余细胞冷冻保存.最后1次实验共分离培养了32个rFNSCs单克隆,其中3个单克隆(9.3%)经PCR、Southern bolt和RT-PCR证实HPRT基因已被敲除.
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