NCKX3 gene disruption in C57BL/6J mice by ZFN (zinc finger nucleases)

Presentation Number: SAT-407
Date of Presentation: June 15th, 2013

Hyun Yang1 and Eui-Bae Jeung*2
1Biochemistry and molecular biology of veterinary medicine college, Chungbuk National Univ, Cheongju, Korea, Republic of (South), 2College of Veterinary Med, Cheongju Chungbuk, Korea, Republic of (South)

Abstract

Gene knockout is the most powerful tool to determine gene function or permanently modify the phenotypic characteristics of animal. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to knockout targeted gene in mice using zinc-finger nucleases (ZFNs). ZFNs are artificial restriction enzymes, designed for induction of double-strand restrictions at a specific gene locus. These double-strand restrictions may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural but imperfect DNA repair process, non-homologous end joining. This process often results in the generation of mutant (knockout) alleles. With the concept of this approach, we designed ZFNs to target the sodium/calcium/potassium exchanger3 (NCKX3) gene in C57bl/6j. We observed heterozygous gene disruption at 25% frequencies, which was sorted by gene sequencing. Seven new genetically distinct NCKX3 +/−mice were generated. The six NCKX3 +/−mice are still alive after delivery without external unusual features. Taken together, we generate NCKX3 +/−mice using ZFN method. Although the mice have not shown any unusual features, the NCKX3 gene function should be further evaluated using the NCKX3 −/−mice.

 

Nothing to Disclose: HY, EBJ