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Mouse mutagenesis Roberta Rivi, MD Laboratory of Molecular Embryology.

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Presentation on theme: "Mouse mutagenesis Roberta Rivi, MD Laboratory of Molecular Embryology."— Presentation transcript:

1 Mouse mutagenesis Roberta Rivi, MD Laboratory of Molecular Embryology

2 Mutagenesis? Goal: determination of the function of every gene (functional genomics)Goal: determination of the function of every gene (functional genomics) Gene-drivenGene-driven –also called reverse genetics Phenotype-drivenPhenotype-driven –also called forward genetics (classical approach)

3 Mutagenesis strategies

4 Insertional mutagens Gene targeting Generates insertions or deletions, as designed. Can design type of mutation as required. Requires knowledge of gene and its structure, labor- intensive, unpredictable phenotypes. TrappingDisrupts endogenous coding sequence. Forward-genetic strategy, easy to clone mutated gene, reports endogenous gene-expression pattern. Unpredictable phenotypes.

5 Homologous recombination HSV-tk HomologyarmHomologyarm

6 Modified from

7 Conditional? Early lethality does not allow to study the role of a gene in organogenesis

8 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

9 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

10 Phage P1: Cre-loxP system

11 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. TrappingDisrupts endogenous coding sequence. Forward-genetic strategy, easy to clone mutated gene, reports endogenous gene-expression pattern. Unpredictable phenotypes.

12 Gene-trap

13 Gene-trap

14 Gene-trap

15 Phenotype driven mutagenesis: chemical mutagenesis Spontaneous 5 x 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 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-Nitroso- Urea 150 x 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.

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17 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.

18 Chemical mutagenesis Modified from RIKEN (The Institute of Physical and Chemical Research)

19 Why Use ENU as a Mutagen Why Use ENU as a Mutagen obtaining a mutation in a single gene of choice in one out of every 175 to 655 gametes screenedENU is an alkylating agent that is a powerful mutagen in mouse spermatogonial stem cells, producing single locus mutation frequencies of 6 X 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.

20 Screening for phenotypes Mapping

21 446.4 unaffected 44.6 affected Line 04/004 LICIA - MATT (e16.5) Smaller embryo Severe microcephaly and micrognathia Abnormal limb morphology & orientation exhibits also a general defect in Endochondral Ossification (all long bones affecteD)

22 Line 04/004 LICIA - MATT Alizarin Red: stains Bone Alcian Blue: stains Cartilage

23 Line 04/014 LICIA - GIUSEPPINA - BETTA mx mb tc unaffected affected mx mb tc hy Rudimentary or hypoplastic mx (maxilla) and mb (mandible - jaw) Absent hyoid Rudimentary tracheal cartilage Alizarin Red: stains Bone Alcian Blue: stains Cartilage

24 Mermaid (merm) Pax3 Snail


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