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

Typical KO vector *tk:thymidine kinase

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

ES cells growing in culture

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

Successfully transformed ES cells are injected into blastocysts

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

Implanting blastocysts 1 2

Implanting blastocysts (cont.) 3 4

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

Chimeric mouse