Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to .1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research. C ONVENTIONAL gene targeting technology in mice relies on homologous recombination in embryonic stem (ES) cells to target specific gene sequences, most commonly to disrupt gene function (Doetschman et al. 1987;Kuehn et al. 1987;Thomas and Capecchi 1987). Advantages of gene targeting in ES cells are selective target sequence modification, the ability to insert or delete genetic information, and the stability of the targeted mutations through subsequent generations. There are also potential limitations, including limited rates of germline transmission and strain limitations due to lack of conventional ES cell lines (Ledermann 2000;Mishina and Sakimura 2007). Moving the targeted allele from one strain to another requires 10 generations of backcrosses that take 2-3 years. A minimum of 1 year is necessary for backcrossing if speed congenics is applied (Markel et al. 1997).Zinc-finger nucleases (ZFNs) are fusions of specific DNA-binding zinc finger proteins (ZFPs) and a nuclease domain, such as the DNA cleavage domain of a type II endonuclease, FokI (Kim et al. 1996;Smith et al. 1999;Bibikova et al. 2001). A pair of ZFPs provide target specificity, and their nuclease domains dimerize to cleave the DNA, generating double strand breaks (DSBs) (Mani et al. 2005), which are detrimental to the cell if left unrepaired (Rich et al. 2000). The cell uses two main pathways to repair DSBs: high-fidelity homologous recombination and error-prone nonhomologous end joining (NHEJ) (Lieber 1999;Pardo et al. 2009;Huertas 2010). ZFN-mediated gene disruption results from deletions or insertions frequently introduced by NHEJ. Figure 1 illustrates the cellular events following the injec...
The rat resembles human physiology more closely than the mouse, partially because of its larger size. Rats are the preferred model for many disease fields, including cancer research, such as studies in breast and prostate cancers and bone metastasis. However, the lack of convenient experimental tools to manipulate the rat genome has largely limited the use of rat models, until the recent creation of the first targeted knockout rats by using zinc finger nuclease technology (Geurts et al., 2009). At SAGE Labs, we are uniquely positioned to create rat knockout models for all major disease categories. In regards to cancer research, we have developed p53 knockout rats, which may be used to significantly shorten the time required for carcinogenicity studies in the same capacity as the p53 knockout mice have been used since the mid-nineties. We are also in the process of creating a suite of immunocompromised rats, such as knockouts of RAG1, RAG2, and DNAPK, which may be used, for example, in the generation of xenografts for metastasis studies and bone marrow transplantation for cancer therapy. In addition, we have developed a set of toxicology models, including knockouts of Mdr1a, PXR, BCRP, Mrp1 and Mrp2, which can benefit studies on cancer drug delivery and metabolism. Model generation, their applications and future models will be discussed. Together, our goal is to help advance cancer research with the creation of novel rat models. Geurts, A. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3235.
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