ENU-Based Gene-Driven Mutagenesis in the Mouse: A Next-Generation Gene-Targeting System

Abstract

Abstract:As a new mouse mutant resource, the RIKEN ENU-based gene-driven mutagenesis system in the mouse has been available to the research community since 2002. By using random base-substitution mutagenesis with ENU, a new reverse genetics infrastructure has been developed as a next-generation gene-targeting system. The construction of a largescale mutant mouse library and high-throughput mutation discovery systems were the keys making it practically feasible. The RIKEN mutant mouse library consists of ~10,000 G1 mice, within which 100-150 mutant strains have been established based on users' requests every year. Use of the system is very simple: users 1) download an application form from our web site and send to us, and 2) design the PCR primers for the target gene. Then, we screen the RIKEN mutant mouse library and report all the detected mutations to the user. From among the allelic series of discovered mutations, users decide which mutant strain(s) to analyze and request the live mutant strain for functional studies of the target gene. Users have been reporting various functional mutations in the RIKEN mutant mouse library: e.g., missense, knockout-type and even functional non-coding mutations. In the near future, next-generation re-sequencing systems should drastically enhance the utility of the ENUbased gene-driven mutagenesis not only for the mouse but also for other species.

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

“…To study the effects of mutations in different domains of TDP‐43 in vivo , at physiological levels, we selected two endogenous Tardbp mutations from a mouse allelic series found by screening mutagenised DNA archives from two large N ‐ethyl‐ N ‐nitrosourea (ENU) mouse mutagenesis programmes (Acevedo‐Arozena et al , 2008; Gondo et al , 2010). The first mutation (F210I) is located in the important RNA recognition motif 2 (RRM2) of TDP‐43; hence, we refer to this mutation as RRM2mut hereafter (Fig 1A).…”

β-cateninC429S mice exhibit sterility consequent to spatiotemporally sustained Wnt signalling in the internal genitalia

“…A high-throughput mutation screening of ENU-mutagenized male genomic DNA library was performed as described previously444546. Four pairs of PCR primers were used for the screening and facilitated the multiplex amplification of exons 3, 5, 8–9 and 11 of the β-catenin gene (Fig.…”

Defective Craniofacial Development and Brain Function in a Mouse Model for Depletion of Intracellular Inositol Synthesis

“…We generated a library of ENU-mutated mice and employed a high throughput screening system to detect mutations. We refer to these techniques collectively as RIKEN ENU-based gene-driven mutagenesis system (RGDMS) (15)(16)(17)(18). Using RGDMS to generate and analyze mice with ENU-induced mutations, we uncovered unexpected functions for Impa1 in the development of the skull, as well as in mouse behavior.…”