ENU

Summary of properties and advantages of ENU mutagenesis

1. ENU is an alkylating agent and has preference for A->T base transversions and also for AT->GC transitions. However it is also shown to cause GC->AT transitions.
2. It is known to induce point mutations, which implies that by mapping for the desired phenotype, the researcher can identify a single candidate gene responsible for the phenotype.
3. The point mutations are at approximately 1-2 Mb (Mega-base-pair) interval and occur at an approximate rate of 1 per 700 gametes.
4. ENU targets spermatogonial stem cells.

ENU - A genetic tool in mutagenesis screens: Overview

Ever since the discovery of ENU as the most potent mutagen by Russell et al. it has been used in forward (phenotype based) genetic screens with which one can identify and study a phenotype of interest. As illustrated in Figure 1, the screening process begins with mutagenising a male mouse with ENU. This is followed by systematic phenotypic analysis of the progeny. The progeny are assessed for behavioral, physiological or dysmorphological changes. The abnormal phenotype is identified. Identification of the candidate gene is then achieved by positional cloning of the mutant mice with the phenotype of interest.

Types of screens

ENU is used as a genetic tool by designing a variety of genetic screens suitable to the researchers' interests. Depending on the region being assessed, forward genetic screens can be classified as illustrated in Figure 2 as:

1. Region Specific screens: Studies are designed specifically to obtain a gradient of phenotypes by generating an allelic series which are helpful in studying the region of interest.
2. Genome-wide screens: These are simple dominant or recessive screens and are often useful in understanding specific genetic and biochemical pathways.

Region-specific screens

Region specific can be classified as follows:

Non-complementation screens

Complementation is the phenomenon which enables generation of the wild type phenotype when organisms carrying recessive mutations in different genes are crossed. Thus if an organism has one functional copy of the gene, then this functional copy is capable of complementing the mutated or the lost copy of the gene. In contrast, if both the copies of the gene are mutated or lost, then this will lead to allelic non-complementation (Figure 3) and thus manifestation of the phenotype.

The phenomenon of redundancy explains that often multiple genes are able to compensate for the loss of a particular gene. However, if two or more genes involved in the same biological processes or pathways are lost, then this leads to non-allelic non-complementation. In a non-complementation screen, an ENU-induced male is crossed with a female carrying a mutant allele (a) of the gene of interest (A). If the mutation is dominant, then it will be present in every generation. However, if the mutation is recessive or if the G₁ progeny are non-viable, then a different strategy is used to identify the mutation. An ENU-treated male is crossed with a wild type female. From the pool of G₁ individuals, a heterozygous male is crossed to a female carrying the mutant allele (a). If the G₂ progeny are infertile or non-viable, they can be recovered again from the G₁ male.

Deletion screens

Deletions on chromosomes can be spontaneous or induced. In this screen, ENU-treated males are crossed to females homozygous for a deletion of the region of interest. The G₁ progeny are compound heterozygotes for the ENU-induced mutation (Figure 4). Also, they are haploid with respect to the genes in the deleted region and thus loss-of-function or gain-of-function due to the ENU-induced mutation is expressed dominantly. Thus deletion screens have an advantage over other recessive screens due to the identification of the mutation in the G₁ progeny itself.

Rinchik et al. performed a deletion screen and complementation analysis and were able to isolate 11 independent recessive loci, which were grouped into seven complementation groups on chromosome 7, a region surrounding the albino (Tyr) gene and the pink-eyed dilution (p) gene.

• c. Balancer screens

A chromosome carrying a balancer region is termed as a balancer chromosome. A balancer is a region which prevents recombination between homologous chromosomes during meiosis. This is possible due to the presence of an inverted region or a series of inversions. Balancer chromosome was primarily used for studies in Drosophila melanogaster genetics. Monica Justice et al. (2009) efficiently carried out a balancer screen using a balancer chromosome constructed by Allan Bradley et al. on mouse chromosome 11. In this screen, an ENU-induced male is crossed with a female heterozygous for the balancer chromosome. The mice carrying the balancer chromosome have yellow ears and tail. The G₁ heterozygotes are (Figure 5) are crossed to females carrying the rex mutation (Rex in figure 5), which confers a curly coat. In G₂, homozygotes for the balancer are non-viable and are not recovered. Mice carrying the rex mutation trans to the balancer or ENU-induced mutation have a curly coat and are discarded. The ENU mutant + rex mutant mice are discarded in order to prevent recombination between those two chromosomes during the next breeding step, which is generating homozygous mutants. Mice that are compound heterozygotes for the balancer and the ENU-induced mutation are brother-sister mated to obtain homozygotes for the ENU-induced mutation in G₃.

Genome-wide screens

Genome-wide screens are most often useful for studying genetic diseases in which multiple genetic and biochemical pathways may be involved. Thus with this approach, candidate genes or regions across the genome, that are associated with the phenotype can be identified.

• a. Conventional screens

These screens can be designed to identify simple dominant and recessive phenotypes. (Figure 6). Thus an ENU-induced G₀ male is crossed with a wild type female. The G1 progeny can be screened to identify dominant mutations. However, if the mutation is recessive, then G₃ individuals homozygous for the mutation can be recovered from the G₁ males in two ways:

• A] The G₁ male is crossed with a wild type female to generate a pool of G₂ progeny. The G₃ individuals can be obtained by crossing the G₁ male to the G₂ daughters. This will yield a proportion of the G₃ individuals which resemble the G₁ male to a large extent.
• B] G₁ male is crossed to a wild type female to obtain a pool of G₂ animals., which are then brother-sister mated to obtain the G₃ progenies. This approach yields a variety of mutants in the G₃ progeny.

A number of organizations around the world are performing genome-wide mutagenesis screens using ENU. Some of them include the Institute of Experimental Genetics at the German Research Center for Environmental Health (GSF), Munich, Germany; The Jackson Laboratory, Maine, USA; the Australian Phenomics Facility at the Australian National University, Canberra, Australia; the Department of Neurobiology and Physiology at Northwestern University, Illinois, USA; the Oak Ridge National Laboratory, Tennessee, USA; the Medical Research Council (MRC) Harwell, Oxfordshire, United Kingdom; the Department of Genetics at The Scripps Research Institute, California, USA; the Mouse Mutagenesis Center for Developmental Defects at Baylor College of Medicine, Texas, USA; and others.

• b. Modifier screens

A modifier such as an enhancer or suppressor can alter the function of a gene. In a modifier screen, an organism with a pre-existing phenotype is selected. Thus, any mutations caused by the mutagen (ENU) can be assessed for their enhancive or suppressive activity. Screening for dominant and recessive mutations is performed in a way similar to the conventional genome-wide screen (Figure 7). A number of modifier screens have been performed on Drosophila. Recently, Aliga et al. performed a dominant modifier screen using ENU-induced mice to identify modifiers of the Notch signaling pathway. Delta 1 is a ligand for the Notch receptor. A homozygous loss-of-function of Delta 1 (Dll1^lacZ/lacZ) is embryonically lethal. ENU-treated mice were crossed to Dll1^lacZ heterozygotes. 35 mutant lines were generated in G₁ of which 7 revealed modifiers of the Notch signaling pathway.

Sensitized screens

In the case of genetic diseases involving multiple genes, mutations in multiple genes contributes to the progression of a disease. Mutation in just one of these genes however, might not contribute significantly to any phenotype. Such "predisposing genes" can be identified using sensitized screens. In this type of a screen, the genetic or environmental background is modified so as to sensitize the mouse to these changes. The idea is that the predisposing genes can be unraveled on a modified genetic or environmental background. Rinchik et al. performed a sensitized screen of mouse mutants predisposed to Diabetic nephropathy. Mice were treated with ENU on a sensitized background of type-1 diabetes. These diabetic mice had a dominant Akita mutation in the insulin-2 gene (Ins2^Akita). These mice developed albuminuria, a phenotype that was not observed in the non-diabetic offsprings.

Stability

Generally speaking, ENU is fairly unstable, which makes it easier to inactivate when used as an experimental mutagen, compared to moderately more stable mutagens like EMS. Pure crystalline ENU is sensitive to light and moisture, so should be stored at in cold and dry conditions, and freshly prepared into solutions when needed. In aqueous solutions, ENU rapidly degrades at a basic pH, and protocols call for inactivation of ENU solutions with an equal volume of 0.1M KOH for 24 hours, with or without ambient light exposure to supplement inactivation.