Mouse genetics: catalogue and scissors

Abstract

Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-Like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics.

Fig. 1.. The basic IKMC knockout allele (tm1a). The lacZ cassette has a 5'-splicing acceptor. Flp removes both lacZ and neo cassettes (tm1c), and Cre deletes both the neo cassette and exon 2 (tm1b). Further action of Cre on tm1c allele removes the critical exon 2 (tm1d). Adopted from Skarnes et al. .

Fig. 2.. The principles of ZFN- or TALEN-mediated gene targeting. (A) Zinc finger (ZF) motifs and (B) TALE modules are designed to detect specific sequences in an opposite direction. Previously developed, heterogeneous nuclease domains of FokI endonuclease can only form a heterodimer that is essential to exert endonuclease activity . (C) When connected to ZFs or TALEs through a linker, each pair of ZFNs or TALENs induces a double-strand break (DSB) at the spacer region between the binding sequences. DSBs are repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). (D) If a donor DNA containing an additional sequence or modifications of the endogenous sequence, HR-mediated gene conversion can be achieved. (E) Without the template for HR-mediated DNA repair, DSB will be repaired through the error-prone NHEJ pathway, frequently resulting in deletion, insertion, and substitution of the original sequence.