Genetics

Genetics

Homologous recombination
1. Specially tailored DNA constructs are introduced into the cell by transfection
  1. A transfected DNA molecule will normally insert into the genome randomly
    1. By including a sequence that is homologous with the target gene, it is possible to make it insert at a predetermined site
  2. The DNA introduced must contain enough sequence homology with its target to insert within at least a few cells in the ES cell culture
    1. The introduced DNA usually carries a drug-resistance gene so that cells carrying the insertion can be selected by adding the drug
      1. Kills the unmodified cells
      2. Mutated Es cells are then introduced into the inner cell mass of a blastocyst , producing a transgenic mouse carrying a mutation in a known gene
        Animals produced by the ES cell transfer are chimeras composed of both mutant and normal cells, and may show few if any effects of the mutation
        If the mutant gene has entered the germline then strains of mice heterozygous for the altered gene can be intercrossed to produce homozygotes that a viable or fail to develop, depending on the gene involved
2. Homologous recombination between the transfected DNA and the target gene in an ES cell results in an insertation that renders the gene non-functional
  1. This is now the main way of producing gene knockouts
    1. When one gene is replaced by another functional gene using these techniques - its called gene knock-in
3. Works particularly well in mice
Gene control elements
Gal4-UAS
1. UAS = Upstream Activation Sequence, an enhancer to which GAL4 specifically binds to activate gene transcription
2. GAL4 = GAL4 gene, encoding the yeast transcription activator protein GAL4
Cre-Lox
GFP and other FPs

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	Created by Kristi Brogden about 10 years ago