This Ptf1aCre-ERTM;KrasLSL-G12D;Ptenflox triple mutant line was generated by crossing three single mutant strains:
C57BL/6J-congenic Ptf1atm2(cre/ESR1)Cvw/J mice from Stock No. 019378,
B6.129S4-Krastm4Tyj/J mice from Stock No. 008179,
B6.129S4-Ptentm1Hwu/J mice from Stock No. 006440.
For the Ptf1atm2(cre/ESR1)Cvw allele [Ptf1aCre-ERTM], targeting vector was designed to replace exons 1-2 of the pancreas specific transcription factor, 1a gene (Ptf1a) with a "loxed cassette acceptor" (LCA) to generate Ptf1aLCA. The LCA contained a reverse-oriented neomycin resistance cassette (neoR) that was flanked by inward-facing loxP sites. Next, a Ptf1aCreERTMHygroR cassette exchange vector was constructed by inserting a 4105 bp fragment of Ptf1a into a plasmid containing two inversely oriented loxP sites, and a 5' UTR NotI site was changed to FseI. DNA encoding Cre-ERTM (Cre-ERT2; Cre recombinase fused to a G525R mutant form of the mouse estrogen receptor ligand binding domain) was then inserted between this FseI and a natural MluI site in Ptf1a exon 1. A pgk-driven hygromycin resistance gene (HygroR), flanked by tandem frt sites, was inserted at the 3' end of the exchange vector for positive selection during recombinase-mediated cassette exchange (RMCE). The Ptf1aCreERTMHygroR cassette exchange vector (along with a cre-expression plasmid to facilitate insertion of the exchange construct) was electroporated into 129S6/SvEvTac-derived TL-1 embryonic stem (ES) cells containing the Ptf1aLCA allele. ES cells, resistant to both neo and hygro, were injected into blastocysts and resulting chimeric mice were bred to C57BL/6J to establish a colony. These mice were subsequently bred to Flp transgenic mice to delete the HygroR cassette, and progeny were crossed to remove the Flp-expressing transgene. The donating investigator reported that these Ptf1aCre-ERTM mice are maintained on a mixed background. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664). The mice were then backcrossed to C57BL/6J inbred mice (Stock No. 000664) for five generations.
For the Krastm4Tyj allele [KrasLSL-G12D], a targeting vector was designed to place a G12D point mutation in exon 1 of the gene and a loxP-flanked STOP element upstream of the mutation. The STOP element incorporates a PGK-puromycin selection cassette at the 5' end in an opposite directional orientation. An adenoviral strong splice acceptor, typically used in gene trap vectors, is fused upstream of the his3 stuffer fragment to prevent splicing around the stopper (in case the transcription isn't completely silenced). A mutant splice donor site is on the 3' end and as well as a tetrameric tandem array of SV40 PolyA. The stopper was designed to fit into genomic Sal1 or Xho1 sites. The stop cassette prevents the expression of mutant Kras until it is removed by Cre mediated recombination of the loxP sites, thus allowing expression of the oncogenic Kras gene. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. This strain was crossed to 129S4/SvJae for more than 10 generations by the donating laboratory which verified correct orientation by Southern assays involving 5' and 3' external and internal probes. The KrasLSL-G12D mice were then backcrossed onto the C57BL/6J background for 14 generations.
For the Ptentm1Hwu allele [Ptenflox], a loxP site flanked targeting vector containing hygromycin resistance and thymidine kinase genes was used in the construction of this mutant. This selection cassette was inserted downstream of exon 5 of the targeted gene, and another loxP site was inserted upstream of exon 5. This construct was electroporated into 129S4/SvJae derived LW-1 embryonic stem (ES) cells, which were transiently transfected with a Cre-recombinase vector to remove the selection cassette. Correctly targeted ES cells were injected into BALB/c blastocysts. The resulting chimeric animals were crossed to BALB/cAnNTac mice before being made homozygous. The Ptenfloxmice were then backcrossed onto the C57BL/6J background for 12 generations.
