1. Vector design Recombinant DNA methods: Simple KO
Structural gene desired (e.g. insulin gene) to be "knocked out" is replaced partly or completely by a positive selection marker. (knock out function!)
Vector DNA to enable the molecules to be inserted into host DNA molecules

2. Typical KO vector
*tk:thymidine kinase

4. Embryonic stem cells
Harvested from the inner cell mass of mouse blastocysts
Grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes

5. ES cells growing in culture

6. ES cells are transformed
Cultured ES cells are exposed to the vector
Electroporation punched holes in the walls of the ES cells
Vector in solution flows into the ES cells
The cells that don't die are selected for transformation using the positive selection marker
Randomly inserted vectors will be killed by gancyclovir

7. Successfully transformed ES cells are injected into blastocysts

8. Implantation of blastocysts
The blastocysts are left to rest for a couple of hours
Expanded blastocysts are transferred to the uterine horn of a 2.5 dpc pseudopregnant female
Max. 1/3 of transferred blasts will develop into healthy pups

11. Implanting blastocysts1 2

12. Implanting blastocysts (cont.)3 4

13. Littermates Black mouse - no apparent ES cell contribution
Chimeric founder -
strong ES cell
contribution
Chimeric founder -
weaker ES cell
contribution

14. Chimeric mouse