Knockout mouse production assisted by Blm knockdown.

The Journal of reproduction and development (2015-11-26)
Mikiko Fukuda, Mayuko Inoue, Daisuke Muramatsu, Hitoshi Miyachi, Yoichi Shinkai
ABSTRACT

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3-4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, clone B-5-1-2, ascites fluid