ENU, also known as N-ethyl-N-nitrosourea (chemical formula C3H7N3O2), is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 gametes. It is also toxic at high doses.
The chemical is an alkylating agent, and acts by transferring the ethyl group of ENU to nucleobases (usually thymine) in nucleic acids. Its main targets are the spermatogonial stem cells, from which mature sperm are derived.
In addition to studying the effect of radiation for SLT, Russell et al. were also interested in studying the effect of chemical mutagens such as procarbazine and ethylnitrosourea for SLT. At that time, procarbazine was the most potent chemical mutagen known to cause a significant spermatogonial mutagenesis in an SLT, although at a rate one-third of that of X-rays. Russell's earlier mutagenesis work on Drosophila using diethylnitrosoamine (DEN) triggered them to use DEN for the SLT. However, DEN needs to be enzymatically converted into an alkylating agent in order to be mutagenic and probably this enzymatic activation was not sufficient in mammals. This could be illustrated by the extremely low mutation rate in mice given by DEN (3 in 60,179 offsprings). To overcome this problem, a new mutagen, N-ethyl N-nitrosourea (ENU), an alkylating agent, which does not need to be metabolised, was suggested to be used by Ekkehart Vegel to Russell et al. The ENU (250 mg/kg) induced mice underwent a period of sterility for 10 weeks. After recovery, 90 males were crossed to the T-stock females and 7584 pups were obtained. Their results showed that a dose of 250 mg/kg of ENU was capable of producing a mutation rate 5 times higher than that obtained with 600R (1R = 2.6 x10^-4 coulombs/kg) of acute X-irradiation. This rate was also 15 times higher to that obtained with procarbazine (600 mg/kg).
To overcome the problem of initial period of sterility, the Russell group showed that instead of injecting one large dose of ENU, a fractionated dose (100mg/kg) on a weekly schedule permitted a total higher dose (300-400mg/kg) to be tolerated. This further showed that the mutation frequency improved to be 12 times that of X-rays, 36 times that of procarbazine and over 200 times that of spontaneous mutations. When the mutation rate was averaged across all 7 loci, ENU was found to induce mutations at a frequency of one per locus in every 700 gametes.
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 many a times 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.
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.
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 primalrily used for studies in Drosophila melanogaster genetics. Monica Justice et al efficiently carried out a balancer screen using a balancer chromosome constructed by Alan Bradley et al on mouse chromosome 11. In this screen, a 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. 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.
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 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.
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 (Dll1lacZ/lacZ) is embryonically lethal. ENU-treated mice were crossed to Dll1lacZ heterozygotes. 35 mutant lines were generated in G1 of which 7 revealed modifiers of the Notch signaling pathway.
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 (Ins2Akita). These mice developed albuminuria, a phenotype that was not observed in the non-diabetic offsprings.