Editing ENU (section)

==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:<ref name=kile/>
# '''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.
# '''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:
[[Image:Non-complementationfig3.JPG|right|thumb|500px|'''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.]]

===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.<ref name=kile/> 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 G<sub>1</sub> 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 G<sub>1</sub> individuals, a heterozygous male is crossed to a female carrying the mutant allele (''a''). If the G<sub>2</sub> progeny are infertile or non-viable, they can be recovered again from the G<sub>1</sub> male.
[[Image:Del.screen.jpg|right|thumb|500px|'''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.]]

===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 G<sub>1</sub> 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 G<sub>1</sub> 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.<ref name=kile/>
[[Image:Balancer screenfig5.JPG|right|thumb|500px|'''Figure 5:''' Balancer Screens.]]
*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.<ref name=kile/> The mice carrying the balancer chromosome have yellow ears and tail. The G<sub>1</sub> heterozygotes are (Figure 5) are crossed to females carrying the rex mutation (''Rex'' in figure 5), which confers a curly coat. In G<sub>2</sub>, 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 G<sub>3</sub>.

===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.
[[Image:Conventional screenfig1.JPG|right|thumb|500px|'''Figure 6:''' Conventional screens.]]
*a. '''Conventional screens'''
These screens can be designed to identify simple dominant and recessive phenotypes. (Figure 6). Thus an ENU-induced G<sub>0</sub> male is crossed with a wild type female. The G<small>1</small> progeny can be screened to identify dominant mutations. However, if the mutation is recessive, then G<sub>3</sub> individuals homozygous for the mutation can be recovered from the G<sub>1</sub> males in two ways:
*A] The G<sub>1</sub> male is crossed with a wild type female to generate a pool of G<sub>2</sub> progeny. The G<sub>3</sub> individuals can be obtained by crossing the G<sub>1</sub> male to the G<sub>2</sub> daughters. This will yield a proportion of the G<sub>3</sub> individuals which resemble the G<sub>1</sub> male to a large extent.
*B] G<sub>1</sub> male is crossed to a wild type female to obtain a pool of G<sub>2</sub> animals., which are then brother-sister mated to obtain the G<sub>3</sub> progenies. This approach yields a variety of mutants in the G<sub>3</sub> 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.<ref name=nolan/>
[[Image:Modifier screenfig7.JPG|right|thumb|500px|'''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.]]
*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.<ref name=kile/> 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.<ref>Rubio-Aliaga, I. et.al. A genetic screen for modifiers of the delta1-dependent notch signaling function in the mouse. Genetics 175, 1451-1463 (2007)</ref> Delta 1 is a ligand for the Notch receptor. A homozygous loss-of-function of Delta 1 (''Dll1<sup>lacZ/lacZ</sup>'') is embryonically lethal. ENU-treated mice were crossed to ''Dll1<sup>lacZ</sup>'' heterozygotes. 35 mutant lines were generated in G<sub>1</sub> 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.<ref>Cordes, S.P. N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express. Microbiol Mol Biol Rev 69, 426-439 (2005).</ref> 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<sup>Akita</sup>''). These mice developed albuminuria, a phenotype that was not observed in the non-diabetic offsprings.<ref>Tchekneva, E.E. et al. A sensitized screen of N-ethyl-N-nitrosourea-mutagenized mice identifies dominant mutants predisposed to diabetic nephropathy. ''J Am Soc Nephrol'' 18, 103-112 (2007).</ref>