Abstract: DNA editing techniques for targeted genome modification have witnessed remarkable advances and been widely used in various organisms. However, traditional gene targeting and cloning method has been shown to be low efficient, time-consuming and expensive for generating knockout animals, especially for big animals. Here we report the generation of site-specific genome modified pig with the newly developed artificially engineered sequence-specific endonucleases (transcription activator-like effector nuclease, TALENs) and handmade cloning (HMC) methods. First, we constructed the porcine GHR-knockout vector according to TALENs kit protocol. To obtain the nuclear donor, the fetal fibroblast cell of Bama (BM) pig were transfected with GHR-knockout vector in G418 selection medium. We collected 173 cell for further positive identification which showed that 46.2% (78/173) of the clones were GHR-knockout cell strains. We chose one bi-allelic knockout cell strain as nuclear donor to produce reconstructed embryos by HMC. It was shown that the blastocyst rate was 43.5% at the 6^th day in vitro, then 654 HMC-blastocysts were transplanted to uterus of six recipient sows. Finally, a total of 10 live offspring were delivered including 7 bi-allelic knockout piglets. Fibroblasts were obtained from ear biopsies for GHR knockout detection. The body weight of the piglets was measured consecutively, and it was found that the GHR^-/- pigs were only 50% smaller than that of the controls at the 20^th week. In conclusion, our results indicate that TALENs and HMC technology can rapidly and efficiently produce knockout animals for agricultural and biomedical research.

Key words: gene knockout, TALENs, handmade cloning, GHR-KO