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

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

Transgenic Analysisneor

5’ fla

nk 3’ flankH

SV-tk Vector for homologousrecombination

Transgenic AnalysisGene of interest

neor

5’ fl

ank 3’ flank

HSV

-tk Vector for homologousrecombination

Transgenic Analysisneor

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.

Transgenic AnalysisneorHSV-tk

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.

Transgenic Analysis

fluoro

iodo

arabinose

Transgenic Analysis

Transgenic Analysis

& FIAU insensitivity

Transgenic Analysis

Transgenic Analysis