1. Functional Analysis of Developmental Genes
Mutant phenotype associated with gene
Generation of mutant phenotype when not already known
Targeted gene disruption (transgenic analysis)
Mis-expression
Ectopic expression
Over expression
Dominant inhibition
Biochemical analysis
Subcellular location
Protein-protein interactions
Enzymology

2. Transgenic Analysis Random insertion of transgenes (for mutagenesis)
Targeted insertion of transgenes
Knockout
Knockin
Requires special vectors
contains flanking sequences to permit homologous recombination between construct and chromosome
Contains selectable marker to permit survival only of homologous recombination and not non-homologous

3. Vector for homologous recombination
Transgenic Analysis
neor
5’ flank
3’ flank
HSV-tk
Vector for homologous recombination

4. Vector for homologous recombination
Transgenic Analysis
Gene of interest
neor
5’ flank
3’ flank
HSV-tk
Vector for homologous recombination

5. Transgenic Analysis
neor
Homologous recombination replaces region of gene with neomycin resistance gene and disrupts generation of functional protein. Neor allows for cells to be selected for using antibiotic neomycin.

6. Transgenic Analysis
HSV-tk
neor
Non-homologous recombination inserts HSV thymidine kinase (tk). The presence of this gene allows cells containing it to be killed by the thymidine analog gancyclovir or FIAU. Only HSV (viral) tk will phosphorylate the nucleotide analog so only the cells with HSV-tk will be killed. The phosphorylated analog inhibits DNA synthesis.

7. Transgenic Analysis
iodo
fluoro
arabinose

8. Transgenic Analysis

9. Transgenic Analysis
& FIAU insensitivity

10. Transgenic Analysis

11. Transgenic Analysis