Genes, Knockouts, and Viruses

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  1. Why would I be interested in a knockout mouse model?
    Maybe you defined the molecular function and want to see the effect of that function

    mouse models are good--> balance of working with models that were really cheap and gets close to the representation of mammalian systems
  2. Producing ES cells containing a targeted gene knockout involves?
    NeoR- provides resistance to neomycin

    Tk+- sensitizes cells to ganciclovir

    NeoR added within exon 2, tk+ added outside exon 2
  3. What are the steps in the creation of the genes?
    Creation of the transgenes involves micropipetting with DNA solution into a single-cell mouse embryo

    The genes will be integrated into the chromosome
  4. Injection of the transgene may result in three products
    1) homologous recombination: chromosome with targeted insertion and NeoR and tk- in the right places; tk+ is lost during homologous recombination

    2) Ectopic (random insertion): a nontarget gene will be inserted into the chromosome

    3) Unchanged chromosome: no neomycin resistance and tk-
  5. How does one select for the correctly recombined cells?
    Selection occurs in neomycin and ganciclovir. Only the homologously recombined cells will be correct
  6. Ganciclovir
    it will kill tk+ cells; becuase tk+ makes it susceptible to Ganciclovir
  7. Where are the cells then inserted into?
    a blastocyst, which gets put into a surrogate mother. A chimeric (transgenic) mouseĀ is eventually born
  8. Explain homologous recombination.
    • it increases variation
    • involved in repair pathways (fixed damaged DNA)
  9. Tk?
    sensitizes a cell to getting circular
  10. Sometimes, we can delete a gene that ____
    leads to an embryonic lethal organism. We don't want to knock out genes in every cell
  11. How do we ensure that we don't knock out every gene in every cell?
    We can control it spatially and temporally.

    • Spatially: What tissue
    • Temporal: time
  12. How can we selectively knock out genes?
    Cre-lox system: We use a Cre recombinase that we exploit to recombine two different regions called lox sites
  13. Explain the Cre-lox system.
    You have two lox sites that flank a gene of interest. Then, Cre recombinase will find these sites, loop them around, cut a piece out to result in the excision of the gene of interest.

    Basically, it cuts out what is between the lox sites.
  14. Cre Mouse and LoxP mouse
    Cre mouse: transgenic mouse that has the Cre recombinase

    LoxP mouse: mouse with loxP sites; a stop signal is before the final loxP site when recombined. The cell will lose the target gene. In this case, GFP is produced
  15. How can we make the tissue specific?
    We make the promoter tissue specific
  16. promoter
    a segment of DNA that regulates expression of that gene
  17. Explain the lac operon.
    • Regulatory gene that makes the repressor protein
    • Promoter region
    • Operator region that binds the repressor protein
    • Genes of interest
  18. repressor protein
    it binds to operator, thus blocking RNA polymerase
  19. By default, what is the lac operon?
    It is off. It results in no RNA production
  20. What happens if lactose is present?
    If lactose is present, it binds the repressor protein, causing a conformational change in the protein and its release from the operator. RNA polymerase is no longer blocked and thus binds to the promoter. Lactose enzymes are produced
  21. We can also induce recombination in what other way?
    by giving it a certain drug

    Ex: Cre can be fused with an estrogen receptor. Regulates the function of Cre with a drug

    Tamoxifen will then affect it
Card Set
Genes, Knockouts, and Viruses
Test Two
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