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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 • Subcellular location • Protein-protein interactions • Enzymology

Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

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Page 1: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

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

Page 2: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

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

Page 3: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysisneor

5’ fl

ank 3’ flank

HSV

-tk

Vector for homologousrecombination

Page 4: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis

Gene of interest

neor

5’ fl

ank 3’ flank

HSV

-tk Vector for homologous

recombination

Page 5: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

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.

Page 6: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis

neorHSV-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.

Page 7: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis

fluoro

iodo

arabinose

Page 8: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis

Page 9: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis

& FIAU insensitivity

Page 10: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis

Page 11: Functional Analysis of Developmental Genes Mutant phenotype associated with gene Generation of mutant phenotype when not already known –Targeted gene disruption

Transgenic Analysis


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