Targeted Gene Modification Of Pig CFTR Gene Using Designed Zinc Finger Nucleases

Cystic fibrosis is an autosomal recessive genetic disease caused by the mutations ofcystic fibrosis transmembrane conductance regulator factor gene. CFTR gene knock-outanimal model produced by ZFN（zinc finger nuclease）technology is important for the CFdisease research and drug screening.The clinical symptoms of CF disease is chronic obstructive pulmonary disease, exocrinepancreatic failure and male infertility, which is treated with small molecule drug clinically.Curcumin is the most studied small molecular activator whcih is the endoplasmic reticulumcalcium pump inhibitors with a low toxic, it can reduce the degradation of CFTR in theprocess after gene mutation, thereby increased the number of membrane chloride ion channeland enhanced cyclic adenosine monophosphate (cAMP)-mediated transmembrane capacity ofchloride ion transport. However, small molecule activators simply reduce the clinicalsymptoms of infection and inflammation by promoting the secretion of chloride ions. Inrecent years, researchers have explored the gene therapy approach for the CF disease,considering security, the vector used to repair the patients’ CFTR gene is difficult for clinicaltreatment. Therefore, the establishment of the CFTR gene knock-out animal models, researchand screening of new drugs for CF disease should be solved indeed.Zinc finger nucleases are novel gene targeted modification technology, which mainlyfocus on gene function research and gene therapies for human diseases, breeding and animalbio-reactors. This technology is able to apply in all species for preciously geneticmanipulation at cellular and individual level. Thus, ZFN technology will be utilized togenerate CF disease swine model, which could play a significant role in CF disease researchand drug screening.In this study, we designed random primers to generate three random3-zinc fingerlibraries via OPEN method, in which the6amino acids in the a helix were randomlymutation. Three zinc finger protein pools with capacity of2×106were obtained to screenspecific zinc finger nucleases. Fifty zinc finger proteins could recognize left and right halftarget site were achieved via bacterial two-hybrid technology through low pressure and highpressure double selection. The selected zinc finger proteins and Fok Ⅰ domain were assembled to generate specific ZFNs.Here, we established a new ZFN activity validation system in yeast cells. In this system,the reporter vector pAC-Gal4-ZFBS contains CFTR target site in the middle of170bp-Gal4BD repeats. In the presence of Gal4BD repeats and target site, Gal4BD has nofunction at all. However, DNA double strand breaks (DSB) generated by ZFNs couldstimulate cellular homologous recombination to repair DSB in yeast cell resulting infunctional Gal4BD and VP16fusion protein. Subsequently, the reporter genes His3and Ade2in yeast strain AH109could express and yeast colonies survive in selective mediums. Thus,ZFNs could cleave target site with highly affinity and specificity were gained.Thus, we used this validation system to investigate the activity of assembled ZFNs afterbacterial two-hybrid system. The results demonstrated that ZFNs could target CFTR site andhomologous recombination event occurred near Gal4BD repeats in the reporter vector togenerate functional Gal4BD-vp16protein. And rate of DNA repairment was about90%.Furthermore, these specific ZFNs will target CFTR gene in swine cells to obtain knock-outcell line, which could be applied to further study on CF disease pathogenesis and drugscreening. In summary, ZFNs obtained through OPEN method and activity validation in yeastcells will be widely used in genome editing.