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Revision as of 17:58, 8 August 2011 editBeetstra (talk | contribs)Edit filter managers, Administrators172,031 edits Script assisted update of identifiers for the Chem/Drugbox validation project (updated: 'ChEBI').← Previous edit Latest revision as of 02:20, 7 September 2024 edit undoBD2412 (talk | contribs)Autopatrolled, IP block exemptions, Administrators2,453,387 editsm Clean up spacing around commas and other punctuation fixes, replaced: ,2002 → , 2002 (2)Tag: AWB 
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{{Other uses|ENU (disambiguation)}} {{other uses|ENU (disambiguation)}}

{{Chembox
{{chembox
⚫ | verifiedrevid = 409133930
| Verifiedfields = changed
⚫ | ImageFile = Ethylnitrosourea.svg
| Watchedfields = changed
⚫ | ImageSize = 180px
⚫ | verifiedrevid = 443717474
⚫ | IUPACName = 1-Ethyl-1-nitrosourea
⚫ | ImageFile = ENU.svg
| OtherNames = Nitrosoethylurea; ''N''-Ethyl-''N''-nitrosourea; ''N''-Ethylnitrosourea
| ImageFile_Ref = {{chemboximage|correct|??}}
⚫ | ImageSize = 140
| ImageFile1 = ENU Ball and Stick.png
| ImageSize1 = 150
| ImageName = Skeletal formula of ENU
⚫ | PIN = ''N''-Ethyl-''N''-nitrosourea
| OtherNames = {{unbulleted list|''N''-Ethylnitrosourea{{citation needed|date=May 2012}}|1-Ethyl-1-nitrosourea|Nitrosoethylurea{{citation needed|date=May 2012}}|''N''-Nitroso-''N''-Ethylurea<ref name=niehs2021>{{cite web |title=N-Nitroso-N-ethylurea |url=https://ntp.niehs.nih.gov/ntp/roc/content/profiles/nitrosamines.pdf |website=Report on Carcinogens, Fourteenth Edition |publisher=NIEHS |access-date=10 August 2021}}</ref>
⚫ }}

| Section1 = {{Chembox Identifiers | Section1 = {{Chembox Identifiers
| Abbreviations = ENU{{citation needed|date=May 2012}}
⚫ | ChemSpiderID_Ref = {{chemspidercite|correct|chemspider}}
⚫ | CASNo = 759-73-9
⚫ | CASNo_Ref = {{cascite|correct|CAS}}
| UNII_Ref = {{fdacite|correct|FDA}}
| UNII = P8M1T4190R
⚫ | PubChem = 12967
| ChemSpiderID = 12427 | ChemSpiderID = 12427
⚫ | ChemSpiderID_Ref = {{chemspidercite|correct|chemspider}}
| InChI = 1/C3H7N3O2/c1-2-6(5-8)3(4)7/h2H2,1H3,(H2,4,7)
| EINECS = 212-072-2
| InChIKey = FUSGACRLAFQQRL-UHFFFAOYAN
| UNNumber = 2811
⚫ | ChEMBL_Ref = {{ebicite|correct|EBI}}
| KEGG = C19178
| KEGG_Ref = {{keggcite|changed|kegg}}
⚫ | ChEBI = 23995
| ChEBI_Ref = {{ebicite|correct|EBI}}
| ChEMBL = 167667 | ChEMBL = 167667
| StdInChI_Ref = {{stdinchicite|correct|chemspider}} | ChEMBL_Ref = {{ebicite|correct|EBI}}
| RTECS = YT3150000
| Beilstein = 1761174
⚫ | SMILES = CCN(N=O)C(N)=O
| StdInChI = 1S/C3H7N3O2/c1-2-6(5-8)3(4)7/h2H2,1H3,(H2,4,7) | StdInChI = 1S/C3H7N3O2/c1-2-6(5-8)3(4)7/h2H2,1H3,(H2,4,7)
| StdInChIKey_Ref = {{stdinchicite|correct|chemspider}} | StdInChI_Ref = {{stdinchicite|correct|chemspider}}
| StdInChIKey = FUSGACRLAFQQRL-UHFFFAOYSA-N | StdInChIKey = FUSGACRLAFQQRL-UHFFFAOYSA-N
| CASNo_Ref = {{cascite|correct|CAS}} | StdInChIKey_Ref = {{stdinchicite|correct|chemspider}}
}}
⚫ | CASNo = 759-73-9

⚫ | PubChem = 12967
⚫ | ChEBI = 23995
⚫ | SMILES = O=C(N)N(N=O)CC
⚫ }}
| Section2 = {{Chembox Properties | Section2 = {{Chembox Properties
| C=3|H=7|N=3|O=2 | C=3 | H=7 | N=3 | O=2
| Appearance = | LogP = 0.208
| Density = | pKa = 12.317
| MeltingPt = | pKb = 1.680
| VaporPressure = 0.00244 kPa @ 25˚C<ref name=niehs2021 />
| BoilingPt =
| Absorbance = ] = 11.86 mM<sup>−1</sup> cm<sup>−1</sup><ref name="CSHP2008">{{cite journal | last1=Salinger | first1=Andrew P. | last2=Justice | first2=Monica J. | title=Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU): Figure 1. | journal=Cold Spring Harbor Protocols | publisher=Cold Spring Harbor Laboratory | volume=2008 | issue=4 | year=2008 | issn=1940-3402 | doi=10.1101/pdb.prot4985 | page=pdb.prot4985| pmid=21356809 | s2cid=1589523 | doi-access=free }}</ref>
| Solubility = }}
}}

| Section3 = {{Chembox Hazards | Section3 = {{Chembox Hazards
| GHSPictograms = {{gHS skull and crossbones}} {{gHS health hazard}}
| MainHazards =
| GHSSignalWord = '''DANGER'''
| FlashPt =
| HPhrases = {{h-phrases|301|312|332|350|360}}
| Autoignition = }}
| PPhrases = {{p-phrases|280|308+313}}
| LD50 = 300 mg kg<sup>−1</sup> <small>(oral, rat)</small>
}}

| Section4 = {{Chembox Related
| OtherFunction_label = ureas
| OtherFunction = {{unbulleted list|]}}
| OtherCompounds = {{unbulleted list|]|]|]|]}}
}}
}} }}


'''ENU''', also known as ''N''-ethyl-''N''-nitrosourea (chemical formula C<sub>3</sub>H<sub>7</sub>N<sub>3</sub>O<sub>2</sub>), is a highly potent ]. For a given ] in ], ENU can induce 1 new ] in every 700 loci. It is also toxic at high doses. '''ENU''', also known as ''N''-ethyl-''N''-]] (chemical formula C<sub>3</sub>H<sub>7</sub>N<sub>3</sub>O<sub>2</sub>), is a highly potent ]. For a given ] in ], ENU can induce 1 new ] in every 700 loci. It is also toxic at high doses.


The chemical is an ] agent, and acts by transferring the ] of ENU to ]s (usually ]) in ]s. Its main targets are the ]s, from which mature ] are derived. The chemical is an ] agent, and acts by transferring the ] of ENU to ]s (usually ]) in ]s. Its main targets are the ]s, from which mature ] are derived.


==Background of discovery of ENU as a mutagen== ==Background of discovery of ENU as a mutagen==
Bill Russell (1951) created a landmark in the field of mouse genetics by creating a specifically designed mouse strain, the ''T'' (test) stock that was used in genetic screens for testing mutagens such as radiations and chemicals. 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 he decided to use ''T''-stock mice in order to define how often a set of loci could be mutated with radiations. Since the mutations in the ''T''-stock mouse were recessive, the progeny would have a wild type phenotype (as a result of crossing a mutant to a wild type female ). 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. This approach, the specific locus test (SLT) allowed Russell to study a wide range of specific mutations and to calculate the mutation rates induced by radiations.<ref name=davis>Davis,A.P., Justice M.J. An Oak Ridge Legacy: The specific locus test and it role in mouse mutagenesis.''Genetics'' 148,7-12 (1998)</ref> Bill Russell (1951) created a landmark in the field of mouse ] by creating a specifically designed mouse strain, the ''T'' (test) stock that was used in genetic screens for testing mutagens such as radiations and chemicals. The ''T''-stock mouse harbors 7 recessive, viable mutations affecting easily recognizable traits. At the ], Russell's initial goal was to determine the rate of inheritable gene mutations in the germ line induced by radiations. Thus he decided to use ''T''-stock mice in order to define how often a set of loci could be mutated with radiations. Since the mutations in the ''T''-stock mouse were ], the progeny would have a wild type ] (as a result of crossing a mutant to a ] female ). 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. This approach, the specific locus test (SLT) allowed Russell to study a wide range of specific mutations and to calculate the mutation rates induced by radiations.<ref name=davis>Davis, A.P., Justice M.J. An Oak Ridge Legacy: The specific locus test and it role in mouse mutagenesis.''Genetics'' 148,7-12 (1998)</ref>


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 ] 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&nbsp;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.<ref name=davis/> Their results showed that a dose of 250&nbsp;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&nbsp;mg/kg).<ref name=russell>Russell W.L., Kelly E.M., Hunsicker P.R., Bangham J.W., Maddux S.C., Phipps E.L. Specific-locus test shows ethylnitrosourea to be the most potent mutagen in mouse. ''Proc. Natl. Acad. Sci.USA'' 11, 5818-5819 (1979)</ref> 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 ] 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 ] 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 offspring). 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&nbsp;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.<ref name=davis/> Their results showed that a dose of 250&nbsp;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&nbsp;mg/kg).<ref name=russell>Russell W.L., Kelly E.M., Hunsicker P.R., Bangham J.W., Maddux S.C., Phipps E.L. Specific-locus test shows ethylnitrosourea to be the most potent mutagen in mouse. ''Proc. Natl. Acad. Sci.USA'' 11, 5818-5819 (1979)</ref>


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 (100&nbsp;mg/kg)<ref name=russell2>Hitotsumachi S., Carpenter D.A., Russell W.L. Dose-Repetition Increases the Mutagenic Effectiveness of N-ethyl-N-nitrosourea in Mouse Spermatogonia. ''Proc. Natl. Acad. Sci.USA'' 82, 6619-6621 (1985)</ref> on a weekly schedule permitted a total higher dose (300–400&nbsp;mg/kg)<ref name=russell2/> 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.<ref name=davis/> 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 (100&nbsp;mg/kg)<ref name=russell2>Hitotsumachi S., Carpenter D.A., Russell W.L. Dose-Repetition Increases the Mutagenic Effectiveness of N-ethyl-N-nitrosourea in Mouse Spermatogonia. ''Proc. Natl. Acad. Sci.USA'' 82, 6619-6621 (1985)</ref> on a weekly schedule permitted a total higher dose (300–400&nbsp;mg/kg)<ref name=russell2/> 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.<ref name=davis/>


==Summary of properties and advantages of ENU mutagenesis== ==Summary of properties and advantages of ENU mutagenesis==
# ENU is an alkylating agent and has preference for A->T base transversions and also for AT->GC transitions.<ref name=nolan>Nolan,P, Hugill, A & Cox,RD,2002,p.278-89</ref> However it is also shown to cause GC->AT transitions.<ref name=coghill>Coghill,EL et al.,2002,p.255-6</ref> # ENU is an alkylating agent and has preference for A->T base transversions and also for AT->GC transitions.<ref name=nolan>Nolan, P, Hugill, A & Cox, RD, 2002, p.278-89</ref> However it is also shown to cause GC->AT transitions.<ref name=coghill>Coghill, EL et al., 2002, p.255-6</ref>
# 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.<ref name=kile>Kile,BT & Hilton, DJ 2005, p.557-67</ref> # 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.<ref name=kile>Kile, BT & Hilton, DJ 2005, p.557-67</ref>
# The point mutations are at approximately 1-2 Mb interval and occur at an approximate rate of 1 per 700 gametes.<ref name=davis/> # The point mutations are at approximately ] interval and occur at an approximate rate of 1 per 700 gametes.<ref name=davis/>
# Point mutations induced by ENU can either be gain-of-function ] or loss-of function mutations in a gene as against deletions, which can induce only loss-of-function mutations.
# ENU targets spermatogonial stem cells.<ref name=nolan/> # ENU targets spermatogonial stem cells.<ref name=nolan/>


] ]


==ENU - A genetic tool in mutagenesis screens: Overview== ==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) ] with which one can identify and study a ] 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 ] of the mutant mice with the phenotype of interest. Ever since the discovery of ENU as the most potent mutagen by Russell et al. it has been used in forward (phenotype based) ] with which one can identify and study a ] 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 ] of the mutant mice with the phenotype of interest.


] ]


==Types of screens== ==Types of screens==
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Depending on the region being assessed, forward genetic screens can be classified as illustrated in Figure 2 as:<ref name=kile/> 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. # '''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 comprise of simple dominant or recessive screens and are often useful in understanding specific genetic and biochemical pathways. # '''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 screens===
Region specific can be classified as follows: Region specific can be classified as follows:
] ]


===Non-complementation screens=== ===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. 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 oftentimes 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. 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. 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.
] ]


===Deletion screens=== ===Deletion screens===
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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/> 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/>
] ]
*c. '''Balancer screens''' *c. '''Balancer screens'''


A chromosome carrying a balancer region is termed as a ]. 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.<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. 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>. A chromosome carrying a balancer region is termed as a ]. 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. ] ''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===
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. 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''' *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: 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:
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*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. *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/> 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/>
] ]
*b. '''Modifier screens''' *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 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. 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=== ===Sensitized screens===
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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> 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>


== Notes == ==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 ]. 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.<ref name=niehs2021 /> 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.<ref name="CSHP2008" />
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== References == ==See also==
* ]
# Kile,BT & Hilton, DJ: The art and design of genetic screens: mouse.''Nat Rev Genet'',6,557-67 (2005)
* ]
# Nolan,P, Hugill, A & Cox,RD: ENU mutagenesis in the mouse: application to human genetic diseases. ''Brief Funct Genomic Proteomic'',1,278-89 (2002)

# Coghill,EL et al.: A gene-driven approach to the identification of ENU mutants in mouse. ''Nat Genet'', 30,255-6 (2002)
==References==
# Cordes, S.P. N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express. Microbiol Mol Biol Rev 69, 426-439 (2005).
⚫ {{reflist|2}}
# Rinchik, E.M., Carpenter, D.A. & Johnson, D.K. Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: a fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15. Proc Natl Acad Sci U S A 99, 844-849 (2002).
# 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).
# 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)
# Davis,A.P., Justice M.J. An Oak Ridge Legacy: The specific locus test and it role in mouse mutagenesis.''Genetics'' 148,7-12 (1998)
# Russell W.L., Kelly E.M., Hunsicker P.R., Bangham J.W., Maddux S.C., Phipps E.L. Specific-locus test shows ethylnitrosourea to be the most potent mutagen in mouse. ''Proc. Natl. Acad. Sci.USA'' 11, 5818-5819 (1979)
# Hitotsumachi S., Carpenter D.A., Russell W.L. Dose-Repetition Increases the Mutagenic Effectiveness of N-ethyl-N-nitrosourea in Mouse Spermatogonia. ''Proc. Natl. Acad. Sci.USA'' 82, 6619-6621 (1985)


==External links== ==External links==
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* * {{Webarchive|url=https://web.archive.org/web/20080910093327/http://www.har.mrc.ac.uk/services/mutagenesis/ |date=2008-09-10 }}
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* * {{Webarchive|url=https://web.archive.org/web/20080614234254/http://www.gsc.riken.go.jp/Mouse/main.htm |date=2008-06-14 }}
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