functional analysis of developmental genes n.
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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

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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
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 analysis1
Transgenic Analysis

neor

5’ flank

3’ flank

Vector for homologousrecombination

HSV-tk

transgenic analysis2

neor

5’ flank

3’ flank

Vector for homologousrecombination

HSV-tk

Transgenic Analysis

Gene of interest

transgenic analysis3
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 analysis4
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 analysis5
Transgenic Analysis

iodo

fluoro

arabinose

transgenic analysis7
Transgenic Analysis

& FIAU insensitivity