Genetic Manipulation Of The Mouse

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An introduction to the techniques of genetic manipulation of the mouse relevant to developmental biology. First presented at CHOP 9/30/2009.

An introduction to the techniques of genetic manipulation of the mouse relevant to developmental biology. First presented at CHOP 9/30/2009.

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  • 1. Genetic Manipulation of the Mouse in Developmental Cardiology Fraz Ahmed Ismat, MD Clinical Associate in Pediatrics The Children’s Hospital of Philadelphia
  • 2. Forward Genetic Screens Embryo & Direct Tissue Genetic Manipulation Manipulation Population Analysis
  • 3. fly mouse zebrafish Forward Genetic frog mouse Screens Embryo & Direct Tissue Genetic Manipulation Manipulation chick chick Population zebrafish Analysis human
  • 4. mouse Direct Genetic Manipulation
  • 5. Types of Genetic Manipulation in Mouse Transgenics Random Insertion Homologous Targeted Recombination Modification
  • 6. Reasons for Genetic Manipulation in Mouse 1) Similarity to People (anatomically, anyway) Global 2) Disruption of Normal Genes Selective 3) “Lineage Tracing”
  • 7. Transgenics in Mouse “foo” promoter “bar” gene
  • 8. Transgenics in Mouse “foo” promoter “bar” gene “foo” domain “bar” expresion
  • 9. Transgenics in Mouse tissue-specific foo? time-specific drug-induced “foo” promoter “bar” gene “bar” “foo” domain expression
  • 10. Transgenics in Mouse tissue-specific GFP LacZ foo? time-specific Gene change bar? drug-induced ?other “foo” promoter “bar” gene “bar” “foo” domain expression
  • 11. Transgenics in Mouse Limitations 1) Limited knowledge of the promoter (NFATc1 enhancer) 2) No regulation of insertion site (CCS-LacZ) 3) What happens to daughter cells?
  • 12. Homologous Recombination I A B C D Genomic Locus Targeting Insert Vector A′ B′ C′ D′ Recombined Insert Locus A B C D
  • 13. Homologous Recombination II Genomic 5′ arm 3′ arm Locus negative 5′ arm Insert neo 3′ arm tox selection positive selection
  • 14. Homologous Recombination II Genomic 5′ arm 3′ arm Locus negative 5′ arm Insert neo 3′ arm tox selection positive selection no insertion death
  • 15. Homologous Recombination II Genomic 5′ arm 3′ arm Locus negative 5′ arm Insert neo 3′ arm tox selection positive selection random insertion no insertion negative selector death death
  • 16. Homologous Recombination II probe probe Genomic 5′ arm 3′ arm Locus negative 5′ arm Insert neo 3′ arm tox selection positive selection random insertion no insertion targeted insertion +neo R -tox negative selector death survival death
  • 17. From Target to Mouse (in only a few years…)
  • 18. From Target to Mouse (in only a few years…) vector es cells
  • 19. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells
  • 20. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells implanted into surrogate
  • 21. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells implanted into chimeric wild type surrogate offspring
  • 22. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells germline! implanted into chimeric wild type surrogate offspring
  • 23. From Target to Mouse (in only a few years…) vector es cells null! blastocyst targeted cells germline! implanted into chimeric wild type surrogate offspring
  • 24. Homologous Recombination III What can the insertion consist of? bar bar bar-null allele bar mutant bar (point mutation, dominant negative, gain-of-function,…) “foo” substitution foo (alternate gene, marker,…) bar “floxed” bar ? what?
  • 25. What is “floxed” for? loxP site “floxed” bar bar “flanked” by loxP sites (i.e. “floxed”) In combination with Cre Recombinase… CONDITIONAL KNOCKOUT!
  • 26. Conditional Knockouts bar foo cre bar
  • 27. Conditional Knockouts bar foo cre bar bar tissue-specific bar time- LacZ “foo” specific “bar” knockout specific bar bar′ drug-induced
  • 28. “Lineage Tracing” (foo-cre × Rosa-LacZ)
  • 29. “Lineage Tracing” (foo-cre × Rosa-LacZ) “foo” expression Daughter cells express LacZ with or without “foo” expression Tissue of also expreses “foo”, interest but outside of our tissue of interest
  • 30. “Lineage Tracing” (foo-LacZ) “foo” expression Daughter cells only express LacZ concurrent with Tissue of “foo” Still interest expressing “foo”
  • 31. Examples of Mouse techniques Transgenics Pax3 NC enahncer Knockout Nf1 knockout(s) “Knock-in” Hop-LacZ Pax3-cre KI Shp2 D61G (Brain, neural crest, CKO Nf1-flox endothelium, SMC, myocardium) “Lineage Tracing” ROSA26-LacZ (+cre lines)
  • 32. Current Problems OVERLAP of two (or more) regions (e.g. Neural crest & secondary heart field) Reliability of current-generation reporters (e.g. ROSA26 vs. GATA4-Flap) TRUE lineage tracing