ENU

The chemical is an alkylating agent, and acts by transferring the ethyl group of ENU to nucleobases (usually thymine) in nucleic acids.

The T-stock mouse harbors 7 recessive, viable mutations affecting easily recognizable traits.

At the Oak Ridge National Laboratory, Russell's initial goal was to determine the rate of inheritable gene mutations in the germ line induced by radiations.

Thus with any progeny carrying a mutation induced by radiation at one of the 7 loci, would exhibit the mutant phenotype in the first generation itself.

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.

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.

[3] 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.

As illustrated in Figure 1, the screening process begins with mutagenising a male mouse with ENU.

Identification of the candidate gene is then achieved by positional cloning of the mutant mice with the phenotype of interest.

Depending on the region being assessed, forward genetic screens can be classified as illustrated in Figure 2 as:[8] Region specific can be classified as follows: Complementation is the phenomenon which enables generation of the wild type phenotype when organisms carrying recessive mutations in different genes are crossed.

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.

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).

From the pool of G1 individuals, a heterozygous male is crossed to a female carrying the mutant allele (a).

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.

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.

Rinchik et al. performed a sensitized screen of mouse mutants predisposed to Diabetic nephropathy.

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.

Skeletal formula of ENU
Skeletal formula of ENU
Figure 1: Overview of ENU mutagenesis screen.
Figure 2: Types of screens.
Figure 3: Non-complementation screens.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.
Figure 4: Deletion Screens.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. 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.
Figure 5: Balancer Screens.
Figure 6: Conventional screens.
Figure 7: Modifier screens.In a modifier screen, an organism with a pre-existing phenotype is selected. Thus the screen is designed to isolate mutants in which the pre-existing phenotype of interest is enhanced or suppressed.