Screening Methods to Identify TALEN-Mediated Knockout Mice

• DOI: 10.1538/expanim.63.79
• Publication Date: 2014-02-06T23:12:32Z
• Authors: Yoshiko Nakagawa; Takashi Yamamoto; Ken-Ichi Suzuki; Kimi Araki; Naoki Takeda; Masaki Ohmuraya; Tetsushi Sakuma
• Keywords: Mice, Knockout; 0303 health sciences; Genome; Genotyping Techniques; Original; Green Fluorescent Proteins; Zinc Fingers; Sequence Analysis, DNA; Mice, Mutant Strains; Mice; 03 medical and health sciences; Genetic Techniques; Mutation; Animals; Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Testing; RNA Editing; Genetic Engineering; Polymorphism, Restriction Fragment Length

Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms.