A study published in Science (2009) describes the successful creation of knockout rats using engineered zinc finger nucleases (ZFNs). The researchers designed ZFNs to target a reporter gene and two endogenous rat genes, IgM and Rab38. Injection of ZFN-encoding DNA or mRNA into one-cell rat embryos resulted in high-frequency mutations at the target locus, with 25–100% disruption in some animals. These mutations were efficiently transmitted through the germline, demonstrating the feasibility of targeted gene disruption in multiple rat strains within four months. This method opens the way for humanized monoclonal antibody platforms and additional human disease models. The rat is a well-established model for studying human disease-related traits, but genome modification has been challenging. ZFNs induce site-specific DNA breaks that can be repaired to create targeted mutations. The study validated ZFN reagents targeting a single-copy GFP transgene and two endogenous genes. Long ZFNs (5- and 6-finger) were used to increase specificity. ZFNs were delivered to embryos via pronuclear or intracytoplasmic injection, resulting in targeted mutations in 12% of founder animals. Full knockout of the GFP transgene was achieved, with 35 animals harboring targeted mutations. Sequence analysis revealed deletions ranging from 3–187 base pairs, and no off-target mutations were detected. Germline transmission was observed for some mutations, with one mutation bred to homozygosity. The study shows that ZFNs can efficiently disrupt genes in rat embryos, offering a valuable tool for rat genetics. This method allows for rapid gene disruption and germline transmission, expanding the potential for human disease modeling. The study was supported by NIH grants and a sponsored research agreement.A study published in Science (2009) describes the successful creation of knockout rats using engineered zinc finger nucleases (ZFNs). The researchers designed ZFNs to target a reporter gene and two endogenous rat genes, IgM and Rab38. Injection of ZFN-encoding DNA or mRNA into one-cell rat embryos resulted in high-frequency mutations at the target locus, with 25–100% disruption in some animals. These mutations were efficiently transmitted through the germline, demonstrating the feasibility of targeted gene disruption in multiple rat strains within four months. This method opens the way for humanized monoclonal antibody platforms and additional human disease models. The rat is a well-established model for studying human disease-related traits, but genome modification has been challenging. ZFNs induce site-specific DNA breaks that can be repaired to create targeted mutations. The study validated ZFN reagents targeting a single-copy GFP transgene and two endogenous genes. Long ZFNs (5- and 6-finger) were used to increase specificity. ZFNs were delivered to embryos via pronuclear or intracytoplasmic injection, resulting in targeted mutations in 12% of founder animals. Full knockout of the GFP transgene was achieved, with 35 animals harboring targeted mutations. Sequence analysis revealed deletions ranging from 3–187 base pairs, and no off-target mutations were detected. Germline transmission was observed for some mutations, with one mutation bred to homozygosity. The study shows that ZFNs can efficiently disrupt genes in rat embryos, offering a valuable tool for rat genetics. This method allows for rapid gene disruption and germline transmission, expanding the potential for human disease modeling. The study was supported by NIH grants and a sponsored research agreement.