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Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion

Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion

Multiple genes involved in cholesterol regulation are required for Andes virus entry. To study hantavirus entry in a biosafety level 2 (BSL-2) setting, we engineered a recombinant vesicular stomatitis virus (rVSV) in which the VSV glycoprotein (G) was replaced with that of Andes virus (ANDV), a prototypic New World hantavirus. Using this agent (rVSV-ANDV GP), we per- formed a loss-of-function genetic screen in haploid human (HAP1) cells, as described previously (22–25). The screen identi- fied seven genes that regulate cellular cholesterol metabolism (Fig. 1A). Four of these genes are critical components of the SREBP (sterol regulatory element-binding protein) cholesterol regulatory pathway (Fig. 1B): (i) SREBF2 (sterol regulatory element-binding transcription factor 2, here termed SREBP2) with 929 disruptive gene trap insertions (see Materials and Meth- ods); (ii) MBTPS1 (membrane-bound transcription factor pepti- dase site 1, here termed site 1 protease [S1P]) with 273 disruptive insertions; (iii) MBTPS2 (membrane-bound transcription factor peptidase site 2, here termed site 2 protease [S2P]) with 218 dis- ruptive insertions; and (iv) SCAP (SREBP cleavage-activating protein) with 142 disruptive insertions. In addition, three more genes encoding enzymes with roles in cholesterol biosynthesis were also identified: (i) LSS (lanosterol synthase) with 55 disrup- tive insertions, (ii) SQLE (squalene epoxidase) with 29 disruptive insertions, and (iii) ACAT2 (acetyl coenzyme A [acetyl-CoA] acetyltransferase 2) with 32 disruptive insertions (Fig. 1B). Most of the gene trap insertions were located toward the 5= end of each gene and enriched for sense orientation insertions, which are more likely to impair gene function due to orientation-dependent
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A Genetic Screen for Modifiers of E2F in Drosophila melanogaster

A Genetic Screen for Modifiers of E2F in Drosophila melanogaster

Other studies have shown that the activity of hSWI/SNF This study demonstrates the use of a genetic screen complexes is itself cell-cycle regulated (Sif et al. 1998). in Drosophila to isolate genes that functionally interact Transformation by activated Ras decreased the expres- with the E2F transcription factor. Four of six genes iden- sion of the murine ortholog of hBRM in mouse fibro- tified are known to regulate gene expression, and three blasts (Muchardt et al. 1998), whereas growth arrest encode components of a chromatin-remodeling com- led to an accumulation of protein (Muchardt et al. plex. These results add to the emerging view that chro- 1998). Recently, BRG1 and BAF155, a human ortholog matin conformation is a key feature of E2F regulation of Moira, were shown to associate with cyclin E and were and suggest that SWI/SNF complexes play an important suggested to be targets for cyclin E-dependent kinases role either in E2F regulation or in the control of S-phase during S-phase entry (Shanahan et al. 1999). entry. We note that this screen is not saturated and that During this study we observed that GMR-dE2FdDP many additional E2F interactors remain to be identified. p35/1; brm 2 /1 eyes developed necrotic patches that
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A Genetic Screen for Hedgehog Targets Involved in the Maintenance of the Drosophila Anteroposterior Compartment Boundary

A Genetic Screen for Hedgehog Targets Involved in the Maintenance of the Drosophila Anteroposterior Compartment Boundary

The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. Here, we report a genetic screen based on the FRT/FLP system to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source we screened 250,000 mutagenized chromosomes. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, we identified a class of patched (ptc) alleles exhibiting a novel phenotype. These results demonstrate the value of our setup in the identification of genes involved in distinct wing-patterning processes.
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Genetic screen identifies a requirement for SMN in mRNA localisation within the Drosophila oocyte

Genetic screen identifies a requirement for SMN in mRNA localisation within the Drosophila oocyte

(Additional file  2: Table  S2). The studies that have thus far explored a role for SMN in oogenesis have been few. Lee et  al. [11] showed that defective nuclear organisa- tion was the most prominent early defect in SMN mutant Drosophila eggs. We have previously observed similar phenotypes in egg chambers mutated for the SMN-asso- ciated DEAD-box helicase, Gemin3 [23, 24]. Considering our assessment of the genetic screen results, we asked whether SMN is also required for the correct localisa- tion of gurken and oskar mRNAs. To this end, we find that in SMN 73Ao mutant oocytes, gurken mRNA was par-
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A Genetic Screen in Drosophila for Identifying Novel Components of the Hedgehog Signaling Pathway

A Genetic Screen in Drosophila for Identifying Novel Components of the Hedgehog Signaling Pathway

along with it the Cos2 complex. These events are corre- These unresolved questions about Hh signaling and lated with the stabilization of full-length Ci-155 and a morphogen movement suggest that additional compo- concomitant loss of repressor Ci-75 (Alcedo et al. 2000; nents to the pathway have yet to be identified. To iden- Denef et al. 2000; Lum et al. 2003). tify novel proteins required for Hh signaling, we con- In wing imaginal discs the derepression of Hh target ducted a large-scale genetic screen in Drosophila. In genes caused by the stabilization of Ci-155 is sufficient this screen we tested the ability of newly induced muta- for the expression of dpp and other low-threshold target tions to enhance or suppress a partial Hh loss-of-func- genes (Methot and Basler 1999). The expression of tion phenotype generated by the transgenic expression high-threshold genes, however, requires the conversion of dominant-negative form of Smo in the developing of Ci-155 into a transcriptional activator and its translo- wing. In addition to new alleles of known components cation into the nucleus (Methot and Basler 2000). of the Hh pathway, 105 interacting mutations, of which This activation of Ci-155 is Hh dependent and requires 34 are grouped into 14 novel complementation groups, Smo protein. The mechanism of Ci-155 activation is were identified. The isolation and genetic characteriza- poorly understood, but likely involves relieving the re- tion of these mutations are described here.
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A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe

A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe

Previously, our lab reported that cells deleted for the N-terminal half of DNA polymerase ε catalytic subunit (Cdc20) are viable, but have a significant S phase delay [25]. Although we expected that this delay would be due to activation of the intra-S phase checkpoint, we were surprised to find that these cells require Chk1, but not Cds1 to maintain cell viability. Our hypothesis was that the Chk1 dependency, Cds1-independency of the DNA polymerase epsilon mutant reflected a unique role for this polymerase in the assembly of the pre-IC. We therefore tested several other DNA replication tempera- ture-sensitive (ts) mutants for their sensitivity to the loss of either Cds1 or Chk1 when grown under semi-permis- sive conditions. Our results suggest that mutants defec- tive in DNA replication initiation (referred to as rid mutants) require the checkpoint kinase Chk1 for viabi- lity, while those defective in the elongation step (referred to as red mutants) require Cds1 [26]. To test our hypothesis further we have now completed a genetic screen for cell division cycle (cdc) mutants that are sen- sitive to the loss of Chk1, but not Cds1. This screen identified novel mutant alleles of cdc30/orp1 and cdc21/
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A Chemical-Genetic Screen for Identifying Substrates of the Er Kinase Perk

A Chemical-Genetic Screen for Identifying Substrates of the Er Kinase Perk

The coming years will undoubtedly see further clarification of PERK signaling mechanisms through identification of additional interactors and downstream targets. From a clinical standpoint, this should provide the opportunity for more selectively targeting the oncogenic potential of PERK, while preserving its vital functions in cellular homeostasis. Prior to this study, there has only been one unbiased screen for PERK substrates (Cullinan et al, 2003). While this screen successfully identified the antioxidant response factor Nrf2, the list of PERK substrates has remained conspicuously small. With the chemical-genetic screen described here, we have identified close to 700 putative PERK substrates. Though this number may seem challenging to prioritize, we have generated substrate lists via two independent methods, which allows concentration on list overlap. Alternatively, it may be prudent to focus mainly on the list of 35
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An Unbiased Genetic Screen Reveals the Polygenic Nature of the Influenza Virus Anti-Interferon Response

An Unbiased Genetic Screen Reveals the Polygenic Nature of the Influenza Virus Anti-Interferon Response

Most of the experimental evidence regarding how influenza virus circumvents the IFN response derives from studies of the pheno- type of NS1 deletion or point mutants and from protein-protein interaction studies involving NS1. In contrast, here we performed an unbiased genetic screen to identify the viral genes that influence the outcome of IFN-mediated innate immune responses to influ- enza virus infections. Instead of increasing the IFN selection pres- sure (65), we carried out serial passage of A/Victoria/3/75 (VIC) influenza virus in non-IFN-responsive cells to release the virus from sequence constraints imposed by the IFN response. A dia- gram showing the experimental strategy employed is presented in Fig. 1. A wt influenza virus was passaged serially in either MDCK or MDCK-V2 cells, which express the V protein of parainfluenza virus type 2 (PIV2) and are thus insensitive to IFN- ␣ / ␤ , since V targets STAT1 for degradation in order to block IFN signaling (51). Cells were infected at a low multiplicity of infection (MOI) (0.001 PFU/cell) to (i) select for efficiently replicating viruses and (ii) prevent the accumulation of defective virus (66). Six indepen- dent serial passages were carried out in parallel to increase the diversity of potential mutants generated.
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A Genetic Screen Reveals that Synthesis of 1,4 Dihydroxy 2 Naphthoate (DHNA), but Not Full Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival

A Genetic Screen Reveals that Synthesis of 1,4 Dihydroxy 2 Naphthoate (DHNA), but Not Full Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival

Although numerous determinants of L. monocytogenes cytosolic replication have been identified (14–16), few determinants of L. monocytogenes cytosolic survival are known (8, 17, 18). Thus, we designed and executed a novel genetic screen to identify L. monocytogenes mutants which lyse in the cytosol of macrophages. We identified mutations in genes regulating central metabolism and genes of unknown function critical for L. monocytogenes survival. Some genes were selectively required for survival in macrophages but not in other cell types, signifying cell type-specific cytosolic defenses. Unexpectedly, through an as-yet-undefined mechanism, a subset of mutants that lyse in the cytosol still avoided inflammasome activation. Despite this, all mutants identified in the screen were attenuated in a murine model of listeriosis. Next we investigated the function of menaquinone (MK) in cytosolic survival. We found that MK’s canonical functions in cellular respiration and the electron transport chain (ETC) were not critical for L. monocytogenes cytosolic survival. Instead, synthesis of the MK biosynthetic intermediate 1,4-dihydroxy-2-naphthoate (DHNA), but not of fully func- tional, isoprenylated menaquinone, was required for L. monocytogenes cytosolic sur- vival. Taking the data together, our genetic screen uncovered factors required for L. monocytogenes survival in the host cytosol and evasion of the innate immune system and ultimately revealed a novel, ETC-independent function for DHNA. Additionally, these results add to the growing body of literature demonstrating that central metab- olism plays a key role during host-pathogen interactions. Not only do host cells monitor and modulate their metabolism to sense and respond to pathogens (19), but cytosolic pathogens must also modulate their metabolism to avoid detection and/or killing by hosts.
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Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

Interestingly, of the original 23 MRB mutant strains, approximately 50% turned out to harbor mutations in laeA or mcsA. Perhaps part of this bias is a result of how the genetic screen was designed, which relied on visual identification of NOR and discarded mutants with extreme developmental phenotypes. This is important for interpretation of these results, as one would expect that a saturated genetic screen would have also discovered additional genes in the velvet complex (VeA and VelB). However, it is understandable that mutations in veA or velB would have been missed in a visual screen due to the presence of increased levels of orsellinic acid produced by these mutants, which makes the strains look very dark in color and masks NOR pigmentation (57). Consistently, deletions of both laeA and mcsA result in loss of ST in addition to other pigments produced by the fungus, thereby making the mutants more easily distin- guishable in a visual NOR screen. Regardless, due to the rediscovery of additional laeA mutants in the screen, we now have more information on which residues are required for proper LaeA function. Two mutations, E190K and W193L, map to the conserved methyltransferase protein domain and may disrupt the protein binding S-adenosyl methionine. The exact role played by the two additional mutated residues (D107 and P330) is unknown as they are located outside the conserved adenosyl methionine (Ado-Met) domain but are highly conserved in other LaeA homologs (see Fig. S3 in the supplemental material). Further study of these mutations may assist in our understand- ing of the mechanistic role that the enigmatic LaeA protein plays in fungal cellular biology.
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Targeted genetic screen in amyotrophic lateral sclerosis reveals novel genetic variants with synergistic effect on clinical phenotype

Targeted genetic screen in amyotrophic lateral sclerosis reveals novel genetic variants with synergistic effect on clinical phenotype

We found that rare deleterious variants in G4C2-repeat-RNA binding partners act synergistically with C9ORF72 expansions to shorten disease duration. This is consistent with work from our group and others providing evidence for sequestration of these proteins by repeat-RNA in C9ORF72-ALS cases (Cooper-Knock et al., 2014, 2015a). Moreover, we identified rare deleterious variants in these proteins in patients without C9ORF72 expansions suggesting that dysfunction of G4C2 binding partners could be pathogenic in the absence of C9ORF72 expansions. Other mechanisms of C9ORF72-ALS pathogenesis have been highlighted in the literature, but our findings support the relative importance of the repeat-RNA sequestration hypothesis. We have shown that, based on proposed RNA toxicity, we could select candidate genes and identify novel ALS genetic variants.
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Targeted Genetic Screen in Amyotrophic Lateral Sclerosis Reveals Novel Genetic Variants with Synergistic Effect on Clinical Phenotype

Targeted Genetic Screen in Amyotrophic Lateral Sclerosis Reveals Novel Genetic Variants with Synergistic Effect on Clinical Phenotype

We tested whether mutations in RNA-binding proteins, including both RRM-containing proteins with a PrLD and G4C2- binding partners, are a cause of ALS and/or whether they modify the clinical phenotype. Our patient cohort (Table 1, Supplementary Table 2) was comprised of either familial ALS cases caused by a C9ORF72 expansion (n = 13) or FALS without a known genetic cause identified (n = 42) or young patients with sporadic ALS (n = 61) who are more likely to carry a pathogenic mutation than older patients with sporadic ALS (Cooper-Knock et al., 2013). Our filtering strategy aimed to identify rare deleterious variants rather than common low-risk variants. We also screened for variants in known ALS genes to augment the analysis and validate our strategy.
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A Genetic Screen for Temperature-Sensitive Cell-Division Mutants of Caenorhabditis elegans

A Genetic Screen for Temperature-Sensitive Cell-Division Mutants of Caenorhabditis elegans

taken place, all three plates were examined for the presence Emb and Stu phenotypes were identified. Eighteen of of progeny males. For several strains, mating could not be these mutations were positioned on the genetic map confirmed in this manner, as mutant hermaphrodites did not using two- and three-factor mapping techniques ( mate- produce live progeny. In these cases, the test was performed

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A Genetic Screen to Identify Components of the sina Signaling Pathway in Drosophila Eye Development

A Genetic Screen to Identify Components of the sina Signaling Pathway in Drosophila Eye Development

URING development of multicellular organisms, cell fate determination is often influenced by in- teractions between neighboring cells. Our understand- ing of the molecular mechanisms underlying such in- teractions has advanced greatly in recent years, and has been aided by a number of model systems. Develop- ment of the R7 photoreceptor in the Drosophila com- pound eye has been a particularly profitable system for dissecting the intercellular signaling pathways used to specify cell fate (reviewed by Wasserman et al . 1995; Simon 1994; Zipursky and Rubin 1994). A large num- ber of genetic, molecular, and biochemical studies have led to identification of many of the signaling compo- nents required for R7 development, their biochemical functions, and their hierarchy within the R7 signaling pathway.
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A Genetic Screen of the Drosophila X Chromosome for Mutations That Modify Deformed Function

A Genetic Screen of the Drosophila X Chromosome for Mutations That Modify Deformed Function

Embryonic lethal Deformed interactors: Mutations in a gene crucial for embryonic Dfd function would be expected to cause defects in or loss of Dfd-dependent cuticular structures, as is seen in Notch, exd, stout, and rnc mutants. As mutations for the other genes identified in our screen do not appreciably affect the formation of the gnathal cuticle, the functions of these genes might be supplied maternally or affect the development of Dfd-dependent tissues not visible in cuticular prepara- tions (e.g., tentorium and subesophageal ganglion). Al- ternatively, some may interact with Dfd only during post- embryonic development. These results are similar to Figure 7.—Gene expression in rnc mutants. (A) Stage 12/
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An Efficient Genetic Screen in Drosophila to Identify Nuclear-Encoded Genes With Mitochondrial Function

An Efficient Genetic Screen in Drosophila to Identify Nuclear-Encoded Genes With Mitochondrial Function

low probability (56%) of localizing to the mitochon- drion. To determine the exact subcellular location of GatA, we constructed a GatA–GFP fusion protein, transfected Drosophila S2 cells with the construct, and co-immunostained the cells with a GFP antibody and MitoTracker Orange CM-H2TMRos, a dye that localizes to the mitochondrion in response to its membrane potential. This colocalization assay shows that GatA is indeed a mitochondrial protein (Figure 4, A–C). Thus, both functional and localization studies and the nature of other mutants isolated by our screen establish that GatA has a mitochondrial function. Five alleles of gatA were isolated from our screen; we have located molec- ular lesions in four alleles (Figure 2). JR94 has a 168-bp deletion in exon 5. JR15 harbors a G . T transversion, resulting in the amino acid substitution Q54H. JR113 has a C . G transversion that causes the substitution H278D. Finally, JV87 has three point mutations, G . T, T . G, and G . A, which result in the following amino acid changes: Q53H, A58S, and V74I. All of the mutations lie within the putative amidase domain of the protein.
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Systematic Genetic Screen for Transcriptional Regulators of the Candida albicans White-Opaque Switch

Systematic Genetic Screen for Transcriptional Regulators of the Candida albicans White-Opaque Switch

An initial screen of the 196 transcriptional regulator mutant strains was carried out to identify regulators with effects on white-to-opaque switching. The white-to-opaque switching rate was calculated by determining the fraction of colonies with one or more opaque sectors for each mutant and nor- malizing it to that observed in wild type colonies grown on the same day (see Materials and Methods for a more detailed de- scription). We could determine white-to-opaque switching rates for 191 of the 196 mutants (see Materials and Methods and File S1 for full explanations of strains examined). Four of these strains exhibited .10-fold increase in white-to-opaque switching (relative to wild type) and 16 strains exhibited a .10-fold reduction in white-to-opaque switching (Figure 2A and Table 1). This list included six of the previously iden- tified regulators of white–opaque switching (Czf1, Efg1, Flo8, Wor1, Wor2, and Wor4) along with 14 previously un- reported regulators, including Gal4, Hap2, Hap31, Hcm1, Mig1, Ndt80, Rap1, and Rpn4. We also identified an addi- tional 11 deletion mutants with less severe effects (five- to 10-fold) on white-to-opaque switching, 10 of which had not previously been linked to switching. Two additional strains
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A Genetic Screen to Identify Genes Required for Drosophila Midline Development

A Genetic Screen to Identify Genes Required for Drosophila Midline Development

Here, we provide evidence that mam and Notch have separate functions in midline glial development. These results suggest that Notch may interact with a co-activator distinct from mam in the AMG. It is possible the bHLH-PAS protein, Sim, could be functioning together with the NICD, either directly or indirectly to activate target genes in the AMG. We have shown that mutating the Sim:Tgo binding site within the regulatory region of wrapper abolished the expression of a wrapper reporter, suggesting that sim directly controls the expression of that gene in the midline glia (Estes et al., 2008). Together, the data suggest a high level of complexity in the regulation of CNS target genes of Notch and that Notch likely interacts with additional cell-lineage specific co-activators other than mam in cells such as midline glia. In this way, modifications of interactors within a signaling pathway can lead to greater cell diversity and function. More broadly, parallel genetic studies in vertebrate systems will facilitate our understanding the developmental roles of mam in both canonical and non-canonical Notch pathways.
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A Genetic Screen for Suppressors of Drosophila NSF2 Neuromuscular Junction Overgrowth

A Genetic Screen for Suppressors of Drosophila NSF2 Neuromuscular Junction Overgrowth

Figure 6.—RT-PCR analysis of gene expression in GS2150 and GS3026. To determine which genes are regulated by the bidirectional GS elements identified in our screen, we used RT-PCR. (A and B) The genomic regions surrounding insertions GS2150 and GS3026, respectively. PCR primers locations are indicated by yellow squares for CG3225 ; by yellow diamonds for polo; by blue diamonds for snap ; by a yellow triangle for CG3793 ; and by green triangles for Gli. The insertion sites for GS3026, GS16634, GS21416, and GS2150 are also shown. “⬍ ⬎” indicates a bidirectional GS insertion; “⬎” represents a unidirectional GS insertion, indicating its orientation. Bar in A applies to B as well. (C) qRT-PCR results show an upregulation of Gli but not of CG3793 by GS2150. The bars represent the amount cDNA induced by Gal4 compared to uninduced samples. (D) GS3026 increases expression of both polo and CG32225. The bar graphs represent the mean and standard error of cDNA levels in four repetitions of hs-Gal4 ⫻ GS line larval extracts relative to GS line alone.
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A Forward Genetic Screen in Mice Identifies Mutants with Abnormal Cortical Patterning

A Forward Genetic Screen in Mice Identifies Mutants with Abnormal Cortical Patterning

Mutagenesis in mice using N-ethyl-N-nitrosourea (ENU) is a means to maximize the efficiency of a phenotype-based forward genetic analysis (Justice et al. 1999; Anderson 2000; Hatten and Heintz 2005; Stottmann and Beier 2010). While identifying the single-base mutations caused by ENU has historically been challenging, the rapid advances in methods of genomic analysis has made positional cloning relatively straightforward (Moran et al. 2006; Fairfield et al. 2011; Leshchiner et al. 2012). Since many human mutations that cause abnormal phenotypes are single-base pair variants, ENU-induced mutations may be a better model of human mutations compared to other gene disruption methods in mice. ENU mutagenesis may help illuminate a function of the specific domain in known genes, as well as the function of novel genes.
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