Genes, Knockouts, and Viruses
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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
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
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
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-
How does one select for the correctly recombined cells?
Selection occurs in neomycin and ganciclovir. Only the homologously recombined cells will be correct
it will kill tk+ cells; becuase tk+ makes it susceptible to Ganciclovir
Where are the cells then inserted into?
a blastocyst, which gets put into a surrogate mother. A chimeric (transgenic) mouse is eventually born
Explain homologous recombination.
- it increases variation
- involved in repair pathways (fixed damaged DNA)
sensitizes a cell to getting circular
Sometimes, we can delete a gene that ____
leads to an embryonic lethal organism. We don't want to knock out genes in every cell
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
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
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.
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
How can we make the tissue specific?
We make the promoter tissue specific
a segment of DNA that regulates expression of that gene
Explain the lac operon.
- Regulatory gene that makes the repressor protein
- Promoter region
- Operator region that binds the repressor protein
- Genes of interest
it binds to operator, thus blocking RNA polymerase
By default, what is the lac operon?
It is off. It results in no RNA production
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
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
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