Construction Of Prnp Gene Knock-Out Vector And Its Transfection In Eukaryotic Cell

Prion protein (Prion protein) is an disease protein of animal transmissible spongiform encephalopathies, a glycoprotein encoded by the gene prnp . Infectious bovine spongiform encephalopathy (BSE) commonly known as mad cow disease, is a serious infectious diseases, harm to human health and the world economic development. A large number of studies have shown that the pathogen of this disease is PrPsc-an endogenous membrane-anchored protein PrPc (coding by prnp genes) isomer, the main event of its incidence process is PrPsc-induced conversion of PrPc to PrPsc. so scientists have inferred the missing animals may have the ability to resist Prion diseases due to lack of the Transformation substrate. this disease has aroused the concern of many of the world and study research group, in which producing a "super cow" resistant to the mad cow disease use of the genetic engineering technology has became one of the largest research hotspot.The key of producing gene transgenic animals by gene targeting is targeting vector. In mammals, the frequency of random reorganization is far higher than homologous recombination, so,in order to improve targeting efficiency, an effective screening stragegy for gene targeting is should be considered. Currently, positive and negative selection stragegy(PNS) is applied more widely, based on this, the study is aimed to construnct a cow prion pryotein gene knockout prnp carrier using Cre-Loxp the principle of recombination system, next, the targeting vector was transfected into bovine fetal fibroblast cells through the electroporation method, then, positive cell clones were screened by PNS strategy and identified through PCR test, immunofluorescence assay, western blotting experiments, finally, gene targeting cells were acquired. The results are as follows:1.Two homologous arms were successfully amplified by PCR from the whole blood genome of cattle, which were 1727bp and 3860bp. Sequencing results showed that the sequence both of short arm and long arm were on line with the sequence published in the GeneBank.2. The bovine prion protein gene knockout vector PBONP was successfully constructed by Inserting the two homologous arms into general-purpose targeting vector PA2T respectively.3.The minimum lethal and maintain concentration of G418 and GCV were established. the minimum lethal and maintain concentration of G418 were 600 ug/mL and 300 ug/mL, the minimum lethal and maintain concentration of GCV were 200 nmol/mL and 100 nmol/mL.4. The targeting vector PBONP was transfected into bovine fetal fibroblast cells through the electroporation method. 176 drug resistant cell clones were acquired through screening by PNS strategy, 9 of which were positive cell clones after identifying through PCR test, immunofluorescence assay, western blotting experiments. Prnp gene deletion bovine fetal fibroblasts was gained.