Zhu Caihong, Yu Guohua, Li Bei, Xu Yuanyuan, Yu Huiqing, Chen Jianquan, Qian Min, Cheng Guoxiang
School of Life Science and Technology, Tongji University, Shanghai 200092, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Nov;24(11):1988-92.
Promoter-trap strategy for enriching targeted colonies has been usually used to elevate the gene targeting efficiency in somatic cells. Knocking out Prnp in animals by gene targeting can render them resistant to Prion diseases. We constructed a bovine Prnp promoter-less targeting vector BoPrPneo, then transfected the linearized vector into the bovine fetal fibroblasts BFF through electroporation. After selecting in cell culture medium with 250 microg/mL G418, we obtained 99 drug-resistant cell colonies, 4 of them were positive for targeted events after PCR screening, and the targeted colonies were further confirmed by sequencing and Southern blotting. This suggests that one allele of Prnp has been successfully knocked out in bovine fetal fibroblasts. This research supplies a simple, safe and effective method to targeting bovine Prnp.
用于富集靶向菌落的启动子捕获策略通常用于提高体细胞中的基因靶向效率。通过基因靶向敲除动物体内的Prnp可使其对朊病毒疾病具有抗性。我们构建了一个无牛Prnp启动子的靶向载体BoPrPneo，然后通过电穿孔将线性化载体转染到牛胎儿成纤维细胞BFF中。在含有250μg/mL G418的细胞培养基中筛选后，我们获得了99个耐药细胞菌落，经PCR筛选其中4个为靶向事件阳性，通过测序和Southern印迹进一步证实了靶向菌落。这表明在牛胎儿成纤维细胞中Prnp的一个等位基因已成功敲除。本研究提供了一种简单、安全且有效的靶向牛Prnp的方法。
