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.

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  • Genetic Manipulation Of The Mouse

    1. 1. Genetic Manipulation of the Mouse in Developmental Cardiology Fraz Ahmed Ismat, MD Clinical Associate in Pediatrics The Children’s Hospital of Philadelphia
    2. 2. Forward Genetic Screens Embryo & Direct Tissue Genetic Manipulation Manipulation Population Analysis
    3. 3. fly mouse zebrafish Forward Genetic frog mouse Screens Embryo & Direct Tissue Genetic Manipulation Manipulation chick chick Population zebrafish Analysis human
    4. 4. mouse Direct Genetic Manipulation
    5. 5. Types of Genetic Manipulation in Mouse Transgenics Random Insertion Homologous Targeted Recombination Modification
    6. 6. Reasons for Genetic Manipulation in Mouse 1) Similarity to People (anatomically, anyway) Global 2) Disruption of Normal Genes Selective 3) “Lineage Tracing”
    7. 7. Transgenics in Mouse “foo” promoter “bar” gene
    8. 8. Transgenics in Mouse “foo” promoter “bar” gene “foo” domain “bar” expresion
    9. 9. Transgenics in Mouse tissue-specific foo? time-specific drug-induced “foo” promoter “bar” gene “bar” “foo” domain expression
    10. 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. 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. 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. 13. Homologous Recombination II Genomic 5′ arm 3′ arm Locus negative 5′ arm Insert neo 3′ arm tox selection positive selection
    14. 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. 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. 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. 17. From Target to Mouse (in only a few years…)
    18. 18. From Target to Mouse (in only a few years…) vector es cells
    19. 19. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells
    20. 20. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells implanted into surrogate
    21. 21. From Target to Mouse (in only a few years…) vector es cells blastocyst targeted cells implanted into chimeric wild type surrogate offspring
    22. 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. 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. 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. 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. 26. Conditional Knockouts bar foo cre bar
    27. 27. Conditional Knockouts bar foo cre bar bar tissue-specific bar time- LacZ “foo” specific “bar” knockout specific bar bar′ drug-induced
    28. 28. “Lineage Tracing” (foo-cre × Rosa-LacZ)
    29. 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. 30. “Lineage Tracing” (foo-LacZ) “foo” expression Daughter cells only express LacZ concurrent with Tissue of “foo” Still interest expressing “foo”
    31. 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. 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

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