B6.Cg-Kras^tm4Tyj Apc^tm1Tno Tg(CDX2-cre/ERT2)752Erf/MaraJ

Stock number: 035169
Product name: KPC:APC
Strain synonyms: KPC:APC, Kras^LSL-G12D;APC^fl;CDX2-Cre-ER^T2
Research areas: Cancer Research, Research Tools

Description

The inducible KPC:APC mouse model of KRAS mutated colorectal cancer combines the oncogenic conditional Kras^G12D allele, the conditional Apc^fl allele and a tamoxifen inducible Cre recombinase (CDX2-Cre-ER^T2) with cre expression in adult epithelium of the distal intestinal tract. Mice develop malignant colonic tumors 25-30 days post tamoxifen induction.

Detailed description

KPC:APC is an inducible mouse model of KRAS mutated colorectal cancer. The model combines 2 mutant alleles and one transgene, each summarized below.
The floxed Kras^G12D allele (Kras^tm4Tyj) carries a G12D point mutation in the Kirsten rat sarcoma viral oncogene homolog (Kras) gene whose expression is blocked by the presence of a loxP-flanked stop codon. Prior to exposure to Cre recombinase, this is a Kras knock-out. In the homozygous state Kras^tm4Tyj is embryonic lethal
The Apc^fl allele (Apc^tm1Tno) has loxP sites flanking exon 14 of the APC, WNT signaling pathway regulator locus. This is a Cre-inducible knock-out allele.
The CDX2-Cre-ER^T2 transgene has a human caudal type homeo box 2 (CDX2) promoter/enhancer sequence directing expression of a tamoxifen-inducible Cre recombinase fusion gene (CreER^T2; a Cre recombinase fused to a human estrogen receptor ligand binding domain) to epithelium of the distal ileum and cecum, and throughout the colon from the crypt base to the luminal surface. While the Cre-ER^T2 is designed to be restricted to the cytoplasm and can only gain access to the nuclear compartment after exposure to tamoxifen, leaky Cre expression from this transgene has been reported (see below).

Mice heterozygous for Kras^tm4Tyj, homozygous for Apc^tm1Tno and hemizygous for CDX2-Cre-ER^T2 are viable and fertile (see breeding considerations under Technical Support).

The donating investigator of the CDX2-Cre-ER^T2 transgene (see Stock No. 022390) reports that cre expression is leaky in 10% of mice. It is the experience of The Jackson Laboratory that cre expression is leaky in approximately 30% of untreated mice as determined by the presence of malignant tumors of the hair follicle (identified as early as 3 weeks of age) in Stock No. 035169 (November 2021).

Following tamoxifen induction, KPC:APC mice develop malignant colonic tumors with within 25-30 days (tamoxifen dose .1 mg/20 g weight). Extensive inflammation with multiple small tumors is observed in the cecum, ascending and transverse colon, and large bowel. Histological and molecular analysis of tumors indicates that they resemble human colorectal carcinoma. Mice live an average of 165 days, dying of cachexia and rectal bleeding. Untreated mice live an average 2 years, 10% of untreated mice develop rectal prolapse around 165 days, subsequently dying of cachexia and rectal bleeding.

Development

For the Kras^tm4Tyj allele, a targeting vector was designed to place a G12D point mutation in exon 1 of the Kras gene on Chromosome 6 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. 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. The resulting chimeric animals were crossed to C57BL/6J and offspring were backcrossed to C57BL/6J for more than 10 generations.

For the Apc^tm1Tno allele (also known as Apc^fl or Apc^580S), the targeting construct contains a loxP site inserted into intron 13 and a PGK neomycin cassette followed by a second loxP site inserted in to intron 14 of the Apc gene on Chromosome 18. Cre-mediated recombination results in deletion of exon 14 resulting in a frameshift silent mutation at codon 580. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were crossed to C57BL/6J and offspring were backcrossed to C57BL/6J for 20 generations.

The CDX2-CreER^T2 transgene was designed with a 9.5 kb promoter fragment from the 5' flanking sequences of human caudal type homeo box 2 (CDX2) gene, a creER^T2 fusion gene (a Cre recombinase fused to a human estrogen receptor ligand binding domain), and a bovine growth hormone polyadenylation cassette. This transgene was injected into (C57BL/6J x SJL/J) hybrid cells. Transgenic mice from founder line 752 were bred to C57BL/6J mice and offspring were crossed to C57BL/6J for at least 10 generations by the donating laboratory (see SNP notes below).

Mice homozygous for Apc^tm1Tno and hemizygous for Tg(CDX2-cre/ERT2)752Erf on the C57BL/6J genetic background were imported from Dr. Radhashree Maitra (Laboratory of Dr. Sanjay Goel, Albert Einstein College of Medicine) and combined with B6.129S4-Kras^tm4Tyj/J (Stock 008179) to establish the triple mutant KPC:APC colony.

In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. One of the 43 markers throughout the genome was segregating on Chromosome 4 with 129. Also, three of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Apc^tm1Tno
Name: targeted mutation 1, Tetsuo Noda
MGI accession ID: MGI:1857966
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Hypomorph
Synonyms: 580S; Apc^{580S flox}; Apc^580s; Apc^f; Apc^fl; Apc^loxP; Apc^lx
Marker symbol: Apc
Marker name: adenomatosis polyposis coli
Marker synonyms: adenomatosis polyposis coli; BTPS2; CC1; DESMD; DP2; DP2.5; DP3; GS; Min; PPP1R46; RATAPC
Chromosome: 18
Strain of origin: 129S4/SvJae
Molecular note: One loxP site was inserted into intron 13 and the other, with a PGK-neo cassette, into intron 14. Northern blot analysis revealed that RNA expression in the intestinal tract of homozygous mutants was about 30% of that in wild-type mice. Protein expression in the colon of homozygotes by immunohistochemistry was indistinguishable from that of wild-type mice. Western blot analysis of lysates from mouse embryo fibroblasts showed reduced expression of the protein.

Allele

Symbol: Kras^tm4Tyj
Name: targeted mutation 4, Tyler Jacks
MGI accession ID: MGI:2429948
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Null/Knockout
Synonyms: caKRas; KR; K-Ras(G12D)^fl; Kras^G12D; K-Ras^L; Kras^Lox; K-ras^LSL; Kras^LSL-G12D; Kras2^tm4Tyj; Kras2tm14Tyj; K-RasG12D; Lox-Stop-Lox-Kras^G12D; LSL-Kras G12D; LSL-K-ras G12D; LSL-Kras^G12D; LSL-K-ras^G12D; LSL-KrasG12D
Marker symbol: Kras
Marker name: Kirsten rat sarcoma viral oncogene homolog
Marker synonyms: CFC2; c-Ki-ras; c-Ki-ras2; 'C-K-RAS; C-K-RAS; Ki-ras; K-ras; K-Ras 2; KRAS1; KRAS2; Kras-2; K-RAS2A; K-RAS2B; K-RAS4A; K-RAS4B; NS; NS3; OES; p21; RALD; RASK2
Chromosome: 6
Strain of origin: 129S4/SvJae
Site of expression: Cre-inducible expression of oncogenic Kras^G12D protein
Molecular note: Glycine codon 12 was changed to aspartic acid (p.G12D) to encode an oncogenic form of the KRAS2 protein. A loxP site flanked STOP cassette was included upstream of the inserted Kras2 sequence, such that the mutant transcript would be expressed only after Cre-mediated recombination.

Allele

Symbol: Tg(CDX2-cre/ERT2)752Erf
Name: transgene insertion 752, Eric Fearon
MGI accession ID: MGI:5477802
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Synonyms: CDX2-creERT2; CDX2P9.5-NLSCre
Marker symbol: Tg(CDX2-cre/ERT2)752Erf
Marker name: transgene insertion 752, Eric Fearon
Chromosome: UN
Strain of origin: (C57BL/6J x SJL/J)
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Upon tamoxifen administration, cre activity was detected in the distal ileal, cecal, colonic, and rectal epithelium.
Molecular note: The transgene was designed with a 9.5 kb promoter fragment from the 5' flanking sequences of human caudal type homeo box 2 (CDX2) gene, a creERT2 fusion gene (a Cre recombinase fused to a human estrogen receptor ligand binding domain), and a bovine growth hormone polyadenylation cassette. This transgene was injected into (C57BL/6J x SJL/J) hybrid cells. Transgenic mice from founder line 752 were bred to C57BL/6J mice. The donating investigator reports that this strain was maintained by breeding hemizygous mice with C57BL/6J inbred mice for at least 4 generations.