Gene targeting in mouse embryonic stem (ES) cells has revolutionized the field of mouse genetics and allowed for the analysis of diverse aspects of gene function in vivo. For more than two decades, researchers have been actively searching for ES cells from large animals such as pigs and cattle. Unfortunately, to date, no ES cell lines from large animals have passed the crucial test of germ line contribution. The sole method of gene targeting to date in these species remains somatic cell nuclear transfer (SCNT) combined with DNA homologous recombination (HR). Due to the limited proliferation competency and extremely low frequency of HR in somatic cells (less than 10−6), this process is highly inefficient and only a few successful examples have been achieved, even though enrichment strategies such as positive-negative marker selection, promoter-trap and adeno-associated viral delivery were previously used 1, 2, 3. The low efficiency of gene targeting in cultured somatic cells is the main barrier for gene targeting in large animals. Recently, zinc-finger nuclease (ZFN) technology has emerged as a powerful tool for genome editing. The success of ZFN technology for gene targeting in fruit flies, zebra fish, rodents as well as human cell lines encouraged us to establish a high-efficiency gene-targeting platform in large animals such as pigs 4, 5, 6, 7, 8.

In zebrafish and rats, methods of direct embryo injection with ZFN-encoding mRNA or plasmids have been employed to generate knockout (KO) mutations 5, 6. This method generates unpredictable deletion/insertion mutations in loci, which commonly result in a mosaic pattern in the first generation of offspring. Further breeding, which is often straightforward in rodents but tedious in large animals, is then necessary to obtain a heritable mutation. In the current study, we combined ZFN technology with SCNT to produce KO pigs with a defined mutation in peroxisome proliferator-activated receptor-gamma (Ppar-γ) (Supplementary information, Data S1). Thiazolidinediones (TZDs), selective ligands of PPAR-γ, are insulin sensitizers clinically used for the treatment of type 2 diabetes 9. Although TZD-mediated activation of PPAR-γ seems to have beneficial effects on cardiovascular diseases (CVD) in mouse models, the therapeutic efficacy of TZDs has been severely compromised because of the increased risk of adverse cardiovascular events in patients 9. As pigs are often considered an ideal animal model for studying human CVD, the Ppar-γ KO pig model will provide an invaluable tool for studying the role of PPAR-γ in CVD.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account
Continue with Google
Continue with ORCiD