摘要
根据已知大鼠次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HypoxanthineGuaninePhosphoribosylTransferase ,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 (BacterialArtificialChromosome ,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 0kb的 5′长臂 (LongArm ,LA)和 1 7kb的 3′短臂 (ShortArm ,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 1μg μl。在FuGene 6转染试剂的作用下转染培养 2 4h的第二代大鼠胎脑神经干细胞 (RatFetalNeuralStemCells,rFNSCs)。转染后的细胞用 80 μg mlG4 18和 0 2 μmol L的Ganc全培养液筛选 ,2w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× 10 3)用裂解液处理 ,取上清用于PCR检测 ,大部分细胞 (5× 10 7)用于DNA和RNA的提取 ,进行Southernbolt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、Southernbolt和RT
The 3.0 kb 5′arm (long arm,LA) of rat HPRT gene knock-out vector was cut by SalⅠ from rat HPRT gene genomic bacterial artificial chromosome (BAC),and the 1.7 kb 3′arm (short arm,SA) was proliferated by PCR.Neo^r,5′arm,3′arm were sequentially cloned into pBS vector's relative restriction enzyme sites.For acquirement of tk gene,the 5′arm -Neo^r-3′arm fragment inserted into pKO vector to construct pKO-HPRT.The pKO-HPRT was linearized by NotⅠ,extracted by ethidium bromide,butanol and phenol/chloroform,and dialyzed by 0.025 μmol/L Millipore.At the same time,rat neural stem cells cultured from E14.5-16.5 rat fetal brain.Passage 2 rFNSCs was tranfected by linearized pKO-HPRT with Fugene-6t transfection reagents.After 80 μg/ml G418 and 0.2 μmol/L ganciclovir selection,the survived cells was cultured in suspension to form neural spheres.The spheres can be picked up under the microscopy,and proliferated in 96-,48- and 24-well plates sequentially.When the cell number reached 4×10~3/well,half cells was lysed by lysis buffer to extract DNA,the other half was kept on growing to freeze and extract RNA.The knock-out cell colonies first detected by PCR,then confirmed by Southern blot and RT-PCR.All the results show that we have knocked out HPRT gene in three rat fetal neural stem cell colonies from 32 colonies.
基金
教育部基金项目 (编号 :0 10 19)~~
