[Construction of prnp gene knockout vector and its transfection in eukaryotic cell].
It is one of the frequently utilized strategies for positive-negative selection to elevate the gene targeting efficiency in somatic cells by enriching targeted colonies. Knocking out prnp in animals by gene targeting can prevent it from expressing Prion protein (Pathogenic protein of transmissible spongiform encephalopathy), which enables it to resist infection of Prion. We constructed a bovine prnp biallelic targeting vector via the positive-negative selection strategy, and transfected the linearized vector into the bovine fetal fibroblasts through electroporation. Then, we selected cells in cell culture medium with G418 under a concentration of 600 microg/mL followed by Ganciclovir (GCV) under a concentration of 200 nmol/mL. In the end, we successfully obtained 176 cell clones. All these clones were identified by means of sequencing, immunofluorescence and western blotting, respectively, confirming that there existed 9 positive cell clones. The results showed that the bovine prnp gene was successfully knocked out. Conclusively, we provide an effective way to knockout bovine prnp gene, which could serve as the basis for producing prion protein gene knockout transgenic cloned cattle.
Key Laboratory of Animal Reproductive Physiology & Embryo Technology, Ministry of Agriculture, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling 712100, China.
Hailin
H
Zhang
Pang
P
Cheng
Jie
J
Lan
Yongli
Y
Song
Yong
Y
Zhang
D000818:Animals; D002417:Cattle; D018274:Electroporation; D016643:Encephalopathy, Bovine Spongiform / Q000235:genetics; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D011328:Prions / Q000235:genetics*; D014162:Transfection
D011328:Prions