Functional Analysis of Developmental Genes

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

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

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