1. 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

2. 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

3. 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

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

5. 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

6. 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

7. 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

8. 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

9. Transgenic Analysis Nucleotide Analogs
iodo
fluoro
arabinose

10. Transgenic Analysis Introduction of Transgene into Cells

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

12. 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:

13. Transgenic Analysis

14. 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

15. Phage Integration/ Recombination
Figure 17.13B 15

16. Cre-Lox Recombination
Figure 17.13A 16

17. Cre-Lox Recombination

19. Ways to Manipulate Floxed Genes