Bulk segregation mapping of mutations in closely related strains of mice.

Department of Genetics, The Scripps Research Institute, La Jolla, California 92037, USA.
Genetics (Impact Factor: 4.87). 10/2010; 186(4):1139-46. DOI: 10.1534/genetics.110.121160
Source: PubMed

ABSTRACT Phenovariance may be obscured when genetic mapping is performed using highly divergent strains, and closely similar strains are preferred if adequate marker density can be established. We sequenced the C57BL/10J mouse genome using the Applied Biosystems SOLiD platform and here describe a genome-wide panel of informative markers that permits the mapping of mutations induced on the closely related C57BL/6J background by outcrossing to C57BL/10J, and backcrossing or intercrossing. The panel consists of 127 single nucleotide polymorphisms validated by capillary sequencing: 124 spaced at ∼20-Mb intervals across the 19 autosomes, and three markers on the X chromosome. We determined the genetic relationship between four C57BL-derived substrains and used the panel to map two N-ethyl-N-nitrosourea (ENU)-induced mutations responsible for visible phenotypes in C57BL/6J mice through bulk segregation analysis. Capillary sequencing, with computation of relative chromatogram peak heights, was used to determine the proportion of alleles from each strain at each marker.


Available from: Xin Du, Jun 03, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses.
    12/2014; 5(4):887-925. DOI:10.3390/genes5040887
  • [Show abstract] [Hide abstract]
    ABSTRACT: We identify an N-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA (PbA). Themis(I23N) homozygote mice show reduced CD4(+) and CD8(+) T lymphocyte numbers. ECM-resistance in PbA-infected Themis(I23N) mice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced pro-inflammatory cytokine production. THEMIS(I23N) protein expression is absent in mutant mice, concurrent with decreased THEMIS(I23N) stability observed in vitro. Biochemical studies in vitro, and functional complementation in vivo in Themis(I23N/+):Lck(-/+) double-heterozygotes demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Dampening of pro-inflammatory responses in Themis(I23N) mice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for development, and ultimately function of pro-inflammatory T cells. Themis(I23N) mice can be used to study the newly discovered association of THEMIS (6p22.33) with inflammatory bowel disease, and multiple sclerosis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Infection and Immunity 12/2014; 83(2). DOI:10.1128/IAI.02586-14 · 4.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: ENU mutagenesis is a forward genetics strategy in which random mutagenesis and phenotypic screening is used to identify genes based on the phenotype induced when they are mutated. A modifier screen is a type of screen in which mice with a pre-existing phenotype are utilized to identify mutations that can enhance or suppress this phenotype. This approach has the potential to uncover missing pathway members, reveal novel genetic interactions, and pinpoint new drug targets. Considerations when planning a suppressor screen include current knowledge, genomic footprint, penetrance, variance, robustness, latency of the starting phenotype, viability, fertility, genetic background and ENU tolerance of starting strain, screening assay, mouse numbers re-quired, and mutation identification strategy. Practical advice on each of these is provided in this review. Curr. Protoc. Mouse Biol. 2:75-87 C 2012 by John Wiley & Sons, Inc.