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Mouse mutagenesis

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).

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Mouse mutagenesis

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  1. Mouse mutagenesis Roberta Rivi, MD Laboratory of Molecular Embryology

  2. 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)

  3. Mutagenesis strategies

  4. Insertional mutagens

  5. HSV-tk Homology arm Homology arm Homologous recombination

  6. Modified from https://cancer.ucsd.edu/tgm/genetargeting.asp

  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 https://bioweb.wku.edu/courses/biol22000/16Recombination/Fig.html

  11. Insertional mutagens

  12. Gene-trap

  13. Gene-trap

  14. Gene-trap

  15. Phenotype driven mutagenesis: chemical mutagenesis

  16. 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.

  17. Chemical mutagenesis Modified from RIKEN (The Institute of Physical and Chemical Research) https://www.gsc.riken.go.jp/Mouse/AboutUs/overview.htm

  18. 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.

  19. Mapping Screening for phenotypes

  20. Line 04/004 LICIA - MATT (e16.5) 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)

  21. Line 04/004 LICIA - MATT 131.5 131.6 Alizarin Red: stains Bone Alcian Blue: stains Cartilage

  22. mx mx mb mb hy tc tc Line 04/014 LICIA - GIUSEPPINA - BETTA unaffected affected 429.1 430.3 Rudimentary or hypoplastic mx (maxilla) and mb (mandible - jaw) Absent hyoid Rudimentary tracheal cartilage Alizarin Red: stains Bone Alcian Blue: stains Cartilage

  23. Pax3 Snail Mermaid (merm)

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