Knockout rats via embryo microinjection of zinc-finger nucleases

Abstract

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.

Fig. 1

ZFN-mediated gene disruption in rat embryos. (A) ZFNs containing 5–6 fingers were designed to target coding sequences of interest (gray lines) for site-specific cleavage. (B) Two of five pups born after microinjection of GFP-targeted ZFNs were devoid of GFP expression. (C) Polymerase chain reaction using GFP-specific primers revealed truncated, but no wild-type sequence in each of the GFP negative pups compared to positive littermates. SS- Dahl S control DNA; NT indicates no template. (D) Table of injection data revealing successful mutagenesis of the three gene targets after multiple delivery methods and doses in three rat strains.