STOCK Kras^tm4Tyj Trp53^tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J

Stock number: 032429
Product name: KPC, tamoxifen-inducible

Research areas: Cancer Research, Research Tools

Description

"KPC, tamoxifen-inducible" is a Kras^LSL-G12D;p53^LoxP;Pdx1-CreER triple mutant model of tamoxifen-inducible pancreatic ductal adenocarcinoma (PDAC).

Detailed description

Stock No. 032429 "KPC, tamoxifen-inducible" is a Kras^LSL-G12D;p53^LoxP;Pdx1-CreER triple mutant model of tamoxifen-inducible pancreatic ductal adenocarcinoma (PDAC) that was assembled at The Jackson Laboratory via combination of three single mutant strains (Stock Nos. 008179, 008462 and 024968, respectively). Each mutant is described in more detail further below.

Using the same three components together along with the Cre-inducible fluorescent reporter allele R26R-Confetti, Maddipati and Stanger 2015 Cancer Discov 5:1086 describes that to induce Cre recombination, tamoxifen was administered to pups via lactation following oral gavage of the mother with 6 mg tamoxifen (suspended in corn oil) on postnatal days 0, 1, 2 and 4. The resulting recombined mice (they called "KPCX") were shown to develop acinar-to-ductal metaplasias and pancreatic intraepithelial neoplasias (PanIN) within 8-10 weeks, and invasive pancreatic ductal adenocarcinoma within 14-16 weeks. They showed metastatic spread to liver, lung, parietal peritoneum and diaphragm. Histological analysis of pancreatic lesions showed multifocal tumors.

The Kras^tm4Tyj allele [Kras^LSL-G12D] contains a point mutation (G12D) whose expression is blocked by the presence of a loxP-flanked stop codon. Mice homozygous for this Kras^tm4Tyj allele die in utero. Cre-mediated recombination can excise the stop codon and permit the oncogenic protein to be expressed.

The Trp53^tm1Brn allele [p53^LoxP] contains loxP sites flanking exons 2 through 10 of the transformation related protein 53 gene. Mice homozygous for the Trp53^tm1Brn allele have a somewhat reduced fertility, but may be bred. Cre-mediated deletion of the floxed sequence results in a knock-out allele.

The Tg(Pdx1-cre/Esr1*)#Dam transgene [Pdx1-CreER] has the mouse Pdx1 promoter driving expression of a tamoxifen-inducible Cre recombinase (Cre-ER^TM; Cre recombinase fused to a G525R mutant form of the mouse estrogen receptor ligand binding domain).
As described for Stock No. 024968, mice homozygous for this Pdx1-CreER transgene are viable and fertile. Immunohistochemistry demonstrates that transgene expression closely follows endogenous Pdx1 patterns. Expression is observed in acini, islet and duct cells but not in mesenchymal cells (endothelial and mesenchyme-derived smooth muscle cells) of the pancreas. Robust Cre expression can be detected by E10.5 in pancreatic epithelium.

Development

This Kras^LSL-G12D;p53^LoxP;Pdx1-CreER triple mutant line was generated by crossing three single mutant strains:
B6.129S4-Kras^tm4Tyj/J Stock No. 008179,
B6.129P2-Trp53^tm1Brn/J Stock No. 008462,
STOCK Tg(Pdx1-cre/Esr1*)#Dam/J Stock No. 024968.

For the Kras^tm4Tyj allele [Kras^LSL-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 the case that transcription isn't completely silenced). A mutant splice donor site is on the 3' end and 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 oncogenic Kras. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. This strain was backcrossed to C57BL/6 for more than 10 generations by the donating laboratory which verified correct orientation by Southern assays involving 5' and 3' external and internal probes.

For the Trp53^tm1Brn mutation [p53^LoxP or p53^flox], a targeting vector was used to introduce flanking loxP sites to introns 1 and 10 of the gene. An IB10/E14IB10 129P2/OlaHsd-derived embryonic stem cell line was used to create the mutation. The mice were then backcrossed to C57BL/6 for eight generations.

For the Tg(Pdx1-cre/Esr1*)#Dam transgene [Pdx1-CreER], a Cre-ER^TM sequence (Cre recombinase fused to a G525R mutant form of the mouse estrogen receptor ligand binding domain) was introduced to the starting ATG of mouse Pdx1, and followed by an SV40 polyadenylation signal. The transgenic vector was injected into B6CBAF1 embryos. Resultant mice were originally crossed with outbred ICR or CD1 animals, then backcrossed to C57BL/6. Subsequent crosses to both ICR/CD1 and C57BL/6 contribute to the STOCK background.

Allele

Symbol: Tg(Pdx1-cre/Esr1*)#Dam
Name: transgene insertion, Douglas A Melton
MGI accession ID: MGI:5008261
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Tg(Pdx1-cre/Esr1*)#Dam
Marker name: transgene insertion, Douglas A Melton
Chromosome: UN
Strain of origin: (C57BL/6 x CBA)F1
Expressed genes: cre/Esr1,Cre recombinase and estrogen receptor 1 fusion gene,
Site of expression: Tamoxifen induction drives mosaic expression of cre recombinase in endocrine, exocrine, and duct tissue of the pancreas.
Molecular note: The sequence encoding a tamoxifen-inducible form of a cre recombinase/estrogen receptor fusion protein was adjoined to the Ipf1 promoter to drive mosaic expression of cre recombinase in endocrine, exocrine, and duct tissue of the pancreas. The pound symbol (#) is used when no line is specified and/or lines are pooled.

Allele

Symbol: Trp53^tm1Brn
Name: targeted mutation 1, Anton Berns
MGI accession ID: MGI:1931011
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Trp53
Marker name: transformation related protein 53
Chromosome: 11
Strain of origin: 129P2/OlaHsd
Molecular note: Insertion of loxP sites flanking exons 2 through 10. No effect on the normal function of the gene.

Allele

Symbol: Kras^tm4Tyj
Name: targeted mutation 4, Tyler Jacks
MGI accession ID: MGI:2429948
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Kras
Marker name: Kirsten rat sarcoma viral oncogene homolog
Chromosome: 6
Strain of origin: 129S4/SvJae
Site of expression: Cre-inducible expression of oncogenic Kras^G12D protein
Molecular note: By homologous recombination in ES cells, the Kras2 locus was targeted with a cassette containing an oncogenic form of the KRAS2 protein in which the glycine at position 12 had been substituted with a an aspartic acid. A loxP flanked stop codon was included upstream of the inserted Kras2 sequence, such that the mutant transcript would be expressed only after cre-mediated recombination.