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
Vector for homologousrecombination
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.
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.
& FIAU insensitivity