Generation of PPARγ knockout pigs by zinc-finger nucleases and nuclear transfer cloning

Diabetes is a major risk factor of cardiovascular disease (CVD). Peroxisome proliferator-activated receptorγ(PPARγ) is a target of the insulin-sensitizingthiazolidinediones (TZDs), a class of drugs widely used for the treatment of type 2 diabetes.The use of experimental rodent models often produces beneficial outcomes of TZDs on heart function that are far different fromobserved heart ischemia risk in human beings. Thus, the conducted mechanisms and results from rodent models cannot be translated to clinical application in terms of beneficial effects of TZDs on the heart. The contrary results may be due to differences in cardiovascular regulation and function between rodents and humans. Therefore, scientists need a new large animal model to mimic the human conditionand distinguish the roles of PPARγ and TZDs in cardiovascular complications associated with diabetes. The pig cardiovascular system, which closely resembles that of humans, is an ideal experimental animal model for studying the regulation of the human cardiovascular system. The research team at University of Michigan led by Dr. Eugene Chen, in collaboration with Dr. Liangxue Lai’s research team at Guangzhou Institutes of Biomedicine and Health (GIBH) in China, successfully generated living PPARγ knock-out piglets, opening a new chapter for studying functions of PPARγ and TZDs in diabetes and cardiovascular complications. This large animal model was obtained by a cutting-edge technology known as zinc-finger nuclease (ZFN). The collaborative research effort was the first touse ZFN technology in order to selectively delete endogenous genes in large animals (Cell Research, 2011 Apr 19).

Due to the lack of established germline-competent embryonic stem (ES) cells, genetargeting in large animalsmight be obtained by a method that combinesDNA homologous recombination with somatic cell nuclear transfer. However,since the proliferation competent of somatic cells is limited and the frequency of homologous recombination in these cells is extremely low (lower than 10-6), this process is highly inefficient and only a few successful examples have been achieved. The ZFN technology has been developed in recent years to generate sequence-specific DNA double-stranded breaks (DSBs) in endogenous genes. ZFNs are fusion proteins in which a zinc-finger DNA-binding domain is linked to the nonspecific endonuclease domain of FokI. The appeal of ZFNs is that their specificity is determined by the zinc finger domain. By altering the specificity of the zinc finger domain, one can alter the target site specificity of the ZFN. This technology has been successfully used for gene targeting in fruit flies, zebra fish, rodents, and human cell lines. In the current study, the efficiency of pig somatic cell gene targeting was dramatically increased (from 10-6 to 4%) using the ZFN technology, which ultimately resulted in the successful generation of two living PPARγ knock-out piglets by nuclear transfer cloning. The research results not only set up a platform to generate piglet models and target endogenous genes with high efficiency, but also offer a reliable approach for gene targeting in large animals that lack ES cells.