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Mouse mutagenesis Roberta Rivi, MD Laboratory of Molecular Embryology Mutagenesis? • Goal: determination of the function of every gene (functional genomics) • Gene-driven – also called reverse genetics • Phenotype-driven – also called forward genetics (classical approach) Mutagenesis strategies Mutagenesis strategy (type) Mutagenesis frequency Spontaneous Primary advantages Primary disadvantages 5 x 1 0-6 per locus Point m utations, small deletions, chromosomal rearrangements, and insertions of endogenous retrovirus-like sequences. Visible phenotypes; only requirement is observant mouse hand lers. Only visi ble pheno types detected, at very l ow frequency. X-ray 13Š50 x 10-5 per locus Chromosomal rearrangements: ranging from simple deletions, inversions and translocations, to complex rearrangements. Rearrangements act as a molecular landmark for cloning. Multiple genes affected, ha rd to dissect individual gene fu nction. Chlorambucil (c hemical mutagen) 127 x10-5 per locus Chromosomal rearrangements, especially s maller deletions (100Š500 kb) and translocations. Same as X-ray, but h igher mutagenesis frequency. Multiple genes affected, ha rd to dissect individual gene fu nction. Ethylnitrosourea (chemical m utagen) 150 x 10-5 per locus Primarily generates point m utations, occasionally very s mall deletions (20Š50 bp). Single-gene mutations, amenable to high thro ughput . No molecular landmarks for cloning. Transgene/ retroviral (insertional mutagen) 5Š10% of transgenic animals Disrupts endogenous gene expression or coding sequence. Sometimes causes chromosomal rearrangements. Provides a molecular landmark for cloning. Labour-intensive, not a pplicable to high-throughp ut approaches. Gene targeting (insertional muta gen) Almost 100% of transgenic animals* Generates insertions or deletions, as designed. Can design type of mutation as required. Requires knowledge of gene and its structure , labour-intensive, unpredictable phenotypes. Trapping (insertional mutagen) Almost 100% of transgenic animals* Disrupts endogenous coding sequence. Forward-genetic strategy, easy to clone muta ted gene, reports endogenous gene-expression pattern. Unpredictable phenotypes. * Requires pre-screening of embryonic stem cells in vitro. Type of mutation induced Insertional mutagens Gene targeting Generates insertions or deletions, as designed. Can design type of mutation as required. Requires knowledge of gene and its structure, laborintensive, unpredictable phenotypes. Trapping Disrupts endogenous coding sequence. Forward-genetic strategy, easy to clone mutated gene, reports endogenous gene-expression pattern. Unpredictable phenotypes. Homologous recombination HSV-tk Homology arm Homology arm Modified from http://cancer.ucsd.edu/tgm/genetargeting.asp Conditional? • Early lethality does not allow to study the role of a gene in organogenesis Conditional? • Early lethality does not allow to study the role of a gene in organogenesis • Tissue-specificity of gene ablation is dependent upon the expression of Cre Conditional? • Early lethality does not allow to study the role of a gene in organogenesis • Tissue-specificity of gene ablation is dependent upon the expression of Cre – One contruct x many Cre lines Phage P1: Cre-loxP system http://bioweb.wku.edu/courses/biol22000/16Recombination/Fig.html Insertional mutagens Gene targeting Generates insertions or deletions, as designed. Can design type of mutation as required. Requires knowledge of gene and its structure, labour-intensive, unpredictable phenotypes. Trapping Disrupts endogenous coding sequence. Forward-genetic strategy, easy to clone mutated gene, reports endogenous gene-expression pattern. Unpredictable phenotypes. Gene-trap Gene-trap Gene-trap Phenotype driven mutagenesis: chemical mutagenesis Spontaneous 5 x 10-6 per locus Point mutations, small deletions, chromosomal rearrangements, and insertions of endogenous retrovirus-like sequences. Visible phenotypes; only requirement is observant mouse handlers. Only visible phenotypes detected, at very low frequency. X-ray 13–50 x 10-5 per locus Chromosomal rearrangements: ranging from simple deletions, inversions and translocations, to complex rearrangements. Rearrangements act as a molecular landmark for cloning. Multiple genes affected, hard to dissect individual gene function. Chlorambucil 127 x10-5 per locus Chromosomal rearrangements, especially smaller deletions (100–500 kb) and translocations. Same as X-ray, but higher mutagenesis frequency. Multiple genes affected, hard to dissect individual gene function. Ethyl-NitrosoUrea 150 x 10-5 per locus Primarily generates point mutations, occasionally very small deletions (20–50 bp). Single-gene mutations, amenable to high throughput. No molecular landmarks for cloning. N-ethyl-N-nitrosourea (ENU) • We are using the chemical N-ethyl-N-nitrosourea (ENU) to saturate wild type chromosomes with point mutations. • By determining the function of genes on a mouse chromosome, we can extrapolate to predict function on a human chromosome. • We expect many of the new mutants to represent models of human diseases such as birth defects, patterning defects, growth and endocrine defects, neurological anomalies, and blood defects. Chemical mutagenesis Modified from RIKEN (The Institute of Physical and Chemical Research) http://www.gsc.riken.go.jp/Mouse/AboutUs/overview.htm Why Use ENU as a Mutagen • ENU is an alkylating agent that is a powerful mutagen in mouse spermatogonial stem cells, producing single locus mutation frequencies of 6 X 10-3 to 1.5 x 10-3, equivalent to obtaining a mutation in a single gene of choice in one out of every 175 to 655 gametes screened. • Because it is a point mutagen, ENU can induce many different types of alleles. Loss of function mutations, viable hypomorphs of lethal complementation groups, antimorphs, and gain-of function mutations have been isolated in mouse mutagenesis screens. • Missense changes are a common finding in many human disease mutations, therefore the ENU mutations will complement and extend the information provided by targeted gene disruptions. Screening for phenotypes Mapping Line 04/004 (e16.5) LICIA - MATT 446.4 unaffected 44.6 affected Smaller embryo Severe microcephaly and micrognathia Abnormal limb morphology & orientation exhibits also a general defect in Endochondral Ossification (all long bones affecteD) Line 04/004 LICIA - MATT 131.5 Alizarin Red: stains Bone Alcian Blue: stains Cartilage 131.6 Line 04/014 LICIA - GIUSEPPINA - BETTA unaffected affected mx mb 429.1 tc Rudimentary or hypoplastic mx (maxilla) and mb (mandible - jaw) Absent hyoid Rudimentary tracheal cartilage Alizarin Red: stains Bone Alcian Blue: stains Cartilage mx 430.3 mb hy tc Pax3 Snail Mermaid (merm)