These triple mutant mice were generated by crossing double mutant mice carrying the Rb1tm2Brn allele and Tg(Pbsn-cre)4Prb transgene to mice carrying the
Akap12tm1Ihg allele.
For the Akap12tm1Ihg allele: a targeting vector containing a lacZ-NEO cassette was used to disrupt exon 2 of the targeted gene. The construct was electroporated into 129S4/SvJae derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into 129SvJ blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were then backcrossed onto the C57BL/6 background for 6 generations. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.
For the Rb1tm2Brn allele: a targeting vector, designed by Dr. Anton Berns (The Netherlands Cancer Institute), was used to introduce loxP sites flanking exon 19 of the targeted gene. The construct was electroporated into unspecified 129 derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were maintained on a mixed FVB;129 background.
For the Tg(Pbsn-cre)4Prb transgene: a transgenic construct, designed by Dr.Pradip Roy-Burman (University of Southern California), containing sequence encoding Cre recombinase under the control of the rat Pbsn, probasin, gene promoter with two tandem repeats of the androgen responsive region, was injected into fertilized (C57BL/6 x DBA/2)F1 mouse eggs. Founder line 1 was subsequently established and maintained on a mixed B6;D2 background.
