Efficient gene targeting by TAL effector nucleases in rats

A research group at Hiroshima University Graduate School of Science, Department of Mathematical and Life Sciences, has successfully generated albino Tyr knockout rats by coinjection of TAL effector nucleases (TALENs) with exonucleases (Exo1) into rat embryos. This approach, combination of exonucleases with engineered TALENs, represents a promising breakthrough for gene targeting technologies applicable to various species, including mice.

Gene targeting technology in experimental animal models has been widely used to study human genetic diseases. Zinc-finger nucleases (ZFNs)/TAL effector nucleases (TALENs) have recently proven to provide a straightforward approach for gene targeting in zygotes, resulting in rapid and effective generation of 'knockout' or 'knockin' animals. Compared with ZFNs, TALENs are simpler to design and can be used in any laboratory that is capable of standard molecular-biochemical procedures. The current limitation of the TALEN technology is not its construction but its efficiency, especially in rats, where only one paper has been published so far compared with more than 10 reports on zebra fish.

Thus, the major goal of this study was to improve the efficiency of TALENs using supportive proteins. In this paper, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species.

To the best of our knowledge, this is the first report to show that certain modifying enzymes could increase the gene targeting efficiency in zygotes by ~5x, thereby improving the gene knockout efficiency in animals. Exo1 mRNA, directly injected with engineered TALENs mRNA into rat embryos, provided an easy, rapid and cost-effective protocol to generate knockout rats.

More information:
Mashimo, T. et al. Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes, Scientific Report, February 13, 2013. dx.doi.org/10.1038/srep01253

Provided by Kyoto University