Transcript Functional Analysis of Developmental Genes

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 Analysis
Vector for homologous
recombination
Transgenic Analysis
Gene of interest
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.
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.
Transgenic Analysis
iodo
arabinose
fluoro
Transgenic Analysis
Transgenic Analysis
& FIAU
insensitivity
Transgenic
Analysis
Transgenic Analysis