利用启动子缺陷型打靶载体敲除牛胎儿成纤维细胞中的Prnp

Targeting Prnp in Bovine Fibroblasts by Promoter-trap Strategy

摘要:

利用启动子捕获对中靶细胞进行富集是提高体细胞基因打靶效率常用的策略之一。敲除动物的Prnp可使其具有抵抗Prion病感染的能力。采用启动子捕获策略, 构建了牛Prnp启动子缺陷型打靶载体BoPrneo, 线性化后, 再通过电穿孔转染牛胎儿成纤维细胞BFF, 用250 mg/mL G418进行药物筛选, 共得到99个药物抗性细胞克隆。对细胞克隆进行PCR、测序及Southern blotting鉴定, 结果表明, 其中的4个细胞克隆为中靶细胞, 说明牛胎儿成纤维细胞中的Prnp一条等位基因被成功敲除。本研究为牛Prnp的敲除提供了一种简单、安全、有效的方法。

Abstract:

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 μg/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.