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Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

Caruana G, Farlie PG, Hart AH, Bagheri-Fam S, Wallace MJ, Dobbie MS, Gordon CT, Miller KA, Whittle B, Abud HE, Arkell RM, Cole TJ, Harley VR, Smyth IM, Bertram JF - PLoS ONE (2013)

Bottom Line: ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads.Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia. georgina.caruana@monash.edu

ABSTRACT

Background: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).

Methodology/principal findings: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.

Conclusions/significance: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

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Related in: MedlinePlus

Lung anomalies displayed by lines 12BCC-013 and 12BCC-016.Lung histology in line 12BCC-013 (A, B) and 12BCC-016 (C, D). Affected lungs in lines 12BCC-013 (B) and 12BCC-016 (D) both displayed hypercellular, thickened lung mesenchyme (m) which can be clearly seen by the increased pink/purple staining of cells and their nuclei, between the small airways (a) in B and D compared to that in the lungs of unaffected littermate embryos (A and C), respectively.
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pone-0055429-g007: Lung anomalies displayed by lines 12BCC-013 and 12BCC-016.Lung histology in line 12BCC-013 (A, B) and 12BCC-016 (C, D). Affected lungs in lines 12BCC-013 (B) and 12BCC-016 (D) both displayed hypercellular, thickened lung mesenchyme (m) which can be clearly seen by the increased pink/purple staining of cells and their nuclei, between the small airways (a) in B and D compared to that in the lungs of unaffected littermate embryos (A and C), respectively.

Mentions: To identify lungs with thick lung mesenchyme a histological screen was undertaken at E18.5 (just prior to birth). We identified two lines 12BCC-013 (7/21 embryos from affected litters, 33%) and 12BCC-016 (3/8 embryos from affected litters, 37.5%) with thick, hypercellular lung mesenchyme (Table 1, Figure 7). These mouse models offered the potential to increase our understanding of the molecular mechanisms that regulate thinning of the lung mesenchyme prior to birth. Unfortunately, the thick hypercellular phenotype was not detected in the second generation mice when searching for carrier males in line 12BCC-013. This may be due to a weakly penetrant lung phenotype or the introduction of a modifier allele that may block the phenotype. Line 12BCC-016 had to be abandoned as the G1 male mouse failed to produce further progeny.


Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

Caruana G, Farlie PG, Hart AH, Bagheri-Fam S, Wallace MJ, Dobbie MS, Gordon CT, Miller KA, Whittle B, Abud HE, Arkell RM, Cole TJ, Harley VR, Smyth IM, Bertram JF - PLoS ONE (2013)

Lung anomalies displayed by lines 12BCC-013 and 12BCC-016.Lung histology in line 12BCC-013 (A, B) and 12BCC-016 (C, D). Affected lungs in lines 12BCC-013 (B) and 12BCC-016 (D) both displayed hypercellular, thickened lung mesenchyme (m) which can be clearly seen by the increased pink/purple staining of cells and their nuclei, between the small airways (a) in B and D compared to that in the lungs of unaffected littermate embryos (A and C), respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585849&req=5

pone-0055429-g007: Lung anomalies displayed by lines 12BCC-013 and 12BCC-016.Lung histology in line 12BCC-013 (A, B) and 12BCC-016 (C, D). Affected lungs in lines 12BCC-013 (B) and 12BCC-016 (D) both displayed hypercellular, thickened lung mesenchyme (m) which can be clearly seen by the increased pink/purple staining of cells and their nuclei, between the small airways (a) in B and D compared to that in the lungs of unaffected littermate embryos (A and C), respectively.
Mentions: To identify lungs with thick lung mesenchyme a histological screen was undertaken at E18.5 (just prior to birth). We identified two lines 12BCC-013 (7/21 embryos from affected litters, 33%) and 12BCC-016 (3/8 embryos from affected litters, 37.5%) with thick, hypercellular lung mesenchyme (Table 1, Figure 7). These mouse models offered the potential to increase our understanding of the molecular mechanisms that regulate thinning of the lung mesenchyme prior to birth. Unfortunately, the thick hypercellular phenotype was not detected in the second generation mice when searching for carrier males in line 12BCC-013. This may be due to a weakly penetrant lung phenotype or the introduction of a modifier allele that may block the phenotype. Line 12BCC-016 had to be abandoned as the G1 male mouse failed to produce further progeny.

Bottom Line: ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads.Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia. georgina.caruana@monash.edu

ABSTRACT

Background: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).

Methodology/principal findings: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.

Conclusions/significance: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

Show MeSH
Related in: MedlinePlus