Functional Analysis of Genes Forward Genetics: Gene associated with mutant phenotype must be identified Reverse Genetics: Generation of mutant phenotype after isolating gene Targeted gene disruption (transgenic analysis) Mis-expression Ectopic expression Over expression Dominant inhibition

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

Cloning of Gene of Interest Cut DNA with restriction enzyme Gene Ligate DNA fragment into vector with ligase enzyme HSV-tk Vector for homologous recombination

Vector for Homologous Recombination Gene 5’ flank 3’ flank HSV-tk

Replace Gene with Selectable Marker Transgene construct neor = neomycin phosphotransferase Such enzymes confer resistance to aminoglycoside antibiotics neomycin G418 kanamycin neor 5’ flank 3’ flank HSV-tk Vector for homologous recombination neo

Transgenic Analysis Homologous Recombination Homologous Chromosomes Gene 1A Ge Gene 1B neor 5’ flank 3’ flank HSV-tk homologous recombination vector Homologous recombination replaces one allele of the gene with the transgene construct Transgene construct

Transgenic Analysis Homologous Recombination Gene 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 or G418. G418 neo

Transgenic Analysis Non-Homologous Recombination HSV-tk neor Non-homologous recombination inserts HSV thymidine kinase (HSVtk). The presence of this gene allows cells containing it to be killed by the thymidine analog gancyclovir or FIAU. The phosphorylated analog inhibits DNA synthesis. Only HSVtk will phosphorylate the nucleotide analog so only the cells with HSVtk will be killed – i.e. only non-homologous recombinants will die – homologous recombinants will live

Transgenic Analysis Nucleotide Analogs iodo fluoro arabinose

Transgenic Analysis Introduction of Transgene into Cells

Transgenic Analysis Recombination in ES Cell Culture Electroporation, Transfection, or Microinjection & FIAU insensitivity

Transgenic Analysis Genotype of transgenic ES cells: bw/bw; BMP7+/- Genotype of host embryo cells: w/w; BMP7+/+ Genotype of chimeras Host derived cells: ES derived cells:

Transgenic Analysis

New Strategies - Floxing Cre/Lox Cre recombinase from phage P1 LoxP site - 34bp site composed of 13bp inverted repeats Construct transgenic organisms with LoxP sites flanking target sequences

Phage Integration/ Recombination Figure 17.13B 15

Cre-Lox Recombination Figure 17.13A 16

Cre-Lox Recombination

Ways to Manipulate Floxed Genes