N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express

Abstract

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.

FIG. 1.

Schematic diagram of basic dominant and recessive schemes in the mouse. After a male G0 mouse of a chosen inbred (such as C57BL/6J) or hybrid strain is treated with ENU, he is bred with normal females. The resulting G1 mice are screened for dominant phenotypes of interest. To perform recessive screens, G3 animals may be generated in several ways. A. In the G2 backcross scheme, G1 males are bred with normal females to produce G2 animals. The original G1 male is crossed with three to six of his G2 daughters, and the resulting G3 animals are examined for the phenotype of interest. B. In the G2 intercross scheme, G2 animals are intercrossed with each other to produce G3 mice for recessive screens.

FIG. 2.

Screening for recessive mutations by using region-specific deletions. In the example shown, which is adapted from reference with permission of the publisher, male mice homozygous for the pink-eyed dilute mutation are treated with ENU and subsequently mated with normal females. The resulting G1 mice are heterozygous for any new mutation, designated by “m,” and the p mutation and are bred with mice compound heterozygous for a deletion spanning the p locus and the pX allele, which gives distinct intermediate eye and coat colors. In the resulting G2 progeny, all normal agouti-colored progeny do not carry a new ENU-induced mutation on this region of chromosome 7. Animals that show the intermediate pink-eyed phenotype are carriers for any new mutations introduced on this chromosome 7 region. All pink-eyed mice will uncover any newly induced, recessive mutations present on the deleted interval.

FIG. 3.

Basic strategy for engineering chromosome-specific deletions and inversions. To generate inversions or deletions, a construct containing a LoxP site and the 5′ half of the puromycin drug resistance gene is inserted either randomly or in a targeted manner into the chromosome in ES cells. A. To create deletions, a construct containing a second LoxP site, the 3′ half of the puromycin gene, and the K14-agouti coat color marker, which causes a yellowish coat color, are introduced either in a targeted manner into a predetermined locus or randomly on the same chromosome. Electroporation of a Cre recombinase-expressing construct results in excision of the sequences flanked by the LoxP site and activation of the puromycin gene. B. To generate inversions, the same construct as was used for generation of deletions is inserted into the chromosome, except this time in the opposite orientation. This time in the presence of Cre recombinase, the puromycin gene is activated but the sequences flanked by LoxP sites are inverted (66).

FIG. 4.

A genetic screen using a “balancer” chromosome in the mouse. A. Normal male mice are treated with ENU and subsequently bred with females heterozygous for a balancer chromosome. In the example shown, the balancer chromosome is marked by the presence of the K14-agouti minigene, which causes a yellowish coat color in mice, and the recessive lethal Wnt3 mutation (Wnt3−). The resulting G1 mice are crossed with animals heterozygous for the balancer and for the dominant Rex mutation, which causes curly hair and helps distinguish the unmutagenized chromosome in the resulting G2 progeny. Curly-haired progeny carry the unmutagenized chromosome and are uninformative. Mice homozygous for the balancer and, thus, also for the Wnt3 mutation die at 10.5 dpc in utero. Mice that have yellowish straight hair are carriers of any mutations that may have been introduced in the inverted region. B. To examine any recessive phenotypes, G2 carrier animals are intercrossed and their progeny analyzed. Once again mice homozygous for the balancer chromosome die at 10.5 dpc; mice with yellowish straight hair are carriers, and agouti (normal)-colored mice represent animals that potentially carry a mutation in the region of interest (28).