Genetic Manipulation by Zinc-Finger Nucleases in Rat-Induced Pluripotent Stem Cells.
Induced pluripotent stem cells (iPSCs) have an extensive application in regenerative medicine, pharmaceutical discovery, and basic research. With the recent derivation of rat iPSCs, it is now feasible to apply genetic manipulation in this species. But such tools do not yet exist for many rat strains, especially for disease model rat. The Sprague Dawley (SD) rat is an inbred disease model for hypertension, nephropathy, pulmonary hypertension, depression, and alcohol consumption. In this study, the SD rat iPSCs were generated using lentiviral method. The p53 gene was targeted in rat iPSCs using homologous recombination mediated by P53 zinc-finger nucleases (ZFNs). Our results showed that these rat iPSCs were pluripotent status. P53 gene was targeted successfully with high efficiency by coelectroporating the donor targeting vectors and p53 ZFN vector to these rat iPSCs. Southern blotting analysis confirmed the correct homologous recombination in rat iPSCs. At the same time, our results demonstrated that the P53 dependent function was abolished in p53-targeted iPSCs. This report also demonstrated that iPS technology, combined with homologous recombination mediated by ZFN, was suitable to develop human disease model in rat and other species.
1 Department of Obstetrics and Gynecology, First Affiliated Hospital of Anhui Medical University , Hefei, P.R. China .|2 The Human Assisted Reproduction Center, Nanchang Institute of Medical Sciences , Nanchang, P.R. China .
Sheng
S
Yang
1 Department of Obstetrics and Gynecology, First Affiliated Hospital of Anhui Medical University , Hefei, P.R. China .|2 The Human Assisted Reproduction Center, Nanchang Institute of Medical Sciences , Nanchang, P.R. China .
Shufang
S
Ding
1 Department of Obstetrics and Gynecology, First Affiliated Hospital of Anhui Medical University , Hefei, P.R. China .
Qianhua
Q
Xu
3 Shanghai JIAI Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University , Shanghai, P.R. China .
Xiong
X
Li
1 Department of Obstetrics and Gynecology, First Affiliated Hospital of Anhui Medical University , Hefei, P.R. China .
Qiong
Q
Xiong
D000818:Animals; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D016086:Lentivirus; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D000075804:Zinc Finger Nucleases / Q000096:biosynthesis / Q000235:genetics
D016159:Tumor Suppressor Protein p53; D000075804:Zinc Finger Nucleases
homologous recombination; induced pluripotent stem cells; p53 gene; zinc-finger nucleases