Genetics

Genetics

1. Homologous recombination

   • Specially tailored DNA constructs are introduced into the cell by transfection
     • A transfected DNA molecule will normally insert into the genome randomly
       • By including a sequence that is homologous with the target gene, it is possible to make it insert at a predetermined site
     • The DNA introduced must contain enough sequence homology with its target to insert within at least a few cells in the ES cell culture
       • The introduced DNA usually carries a drug-resistance gene so that cells carrying the insertion can be selected by adding the drug
         • Kills the unmodified cells
         • 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
   • Homologous recombination between the transfected DNA and the target gene in an ES cell results in an insertation that renders the gene non-functional
     • This is now the main way of producing gene knockouts
       • When one gene is replaced by another functional gene using these techniques - its called gene knock-in
   • Works particularly well in mice

2. Gene control elements

3. Gal4-UAS

   • UAS = Upstream Activation Sequence, an enhancer to which GAL4 specifically binds to activate gene transcription
   • GAL4 = GAL4 gene, encoding the yeast transcription activator protein GAL4

4. Cre-Lox

5. GFP and other FPs