An Msh2 conditional knockout mouse for studying intestinal cancer and testing anticancer agents

Abstract

Background & aims: Mutations in the DNA mismatch repair (MMR) gene MSH2 cause Lynch syndromes I and II and sporadic colorectal cancers. Msh2(null) mice predominantly develop lymphoma and do not accurately recapitulate the colorectal cancer phenotype.

Methods: We generated and examined mice with a conditional Msh2 disruption (Msh2(LoxP)), permitting tissue-specific gene inactivation. ECMsh2(LoxP/LoxP) mice carried an EIIa-Cre transgene, and VCMsh2(LoxP/LoxP) mice carried a Villin-Cre transgene. We combined the VCMsh2(LoxP) allele with either Msh2(Delta7null) (VCMsh2(LoxP/null)) or Msh2(G674D) mutations (VCMsh2(LoxP/G674D)) to create allelic phase mutants. These mice were given cisplatin or 5-fluorouracil/leucovorin and oxaliplatin (FOLFOX), and their tumors were measured by magnetic resonance imaging.

Results: Embryonic fibroblasts from ECMsh2(LoxP/LoxP) mice do not express MSH2 and are MMR deficient. Reverse transcription, polymerase chain reaction, and immunohistochemistry from VCMsh2(LoxP/LoxP) mice demonstrated specific loss of Msh2 messenger RNA and protein from epithelial cells of the intestinal tract. Microsatellite instability was observed in all VCMsh2 strains and limited to the intestinal mucosa. Resulting adenomas and adenocarcinomas had somatic truncation mutations to the adenomatous polyposis coli (Apc) gene. VCMsh2(LoxP/LoxP) mice did not develop lymphoma. Comparison of allelic phase tumors revealed significant differences in multiplicity and size. When treated with cisplatin or FOLFOX, tumor size was reduced in VCMsh2(LoxP/G674D) but not VCMsh2(LoxP/null) tumors. The apoptotic response to FOLFOX was partially sustained in the intestinal mucosa of VCMsh2(LoxP/G674D) animals.

Conclusions: Msh2(LoxP/LoxP) mice in combination with appropriate Cre recombinase transgenes have excellent potential for preclinical modeling of Lynch syndrome, MMR-deficient tumors of other tissue types, and use in drug development.

Figure 1

Strategy for the production of Msh2 conditional knockout mutant mice, and PCR genotyping of offspring. (A) Gene targeting strategy. (B) PCR genotyping strategy. 1) pGEM markers, 2) Wildtype mouse, 3) Msh2^LoxP/LoxP, exon 12 deleted, 4) Msh2^LoxP/+. (C) Where Msh2 exon 12 has been deleted, primer pair 184F/184R amplifies no product, whereas primer pair 184F/165R amplifies a 340 bp product. Wildtype animals without the conditional allele amplify a 210 bp PCR product using primers 184F/184R.

Figure 2

Molecular characterization of VCMsh2^LoxP/LoxP and ECMsh2^LoxP/LoxP mice. (A) Specific rearrangement of the Msh2 gene in the small and large intestines of VCMsh2^LoxP/LoxP animals, is absent in the kidney heart and spleen. Primer pairs A/C amplify a 340 bp product when exon 12 is deleted (intestine) and a 983 bp product when exon 12 is intact (kidney heart and spleen). (B) Measurement of MMR in ECMsh2^LoxP/LoxP MEFs. Cell lines EM2-1 and EM2-2 are MMR deficient and compare to the Exo1^-/- MMR deficient cell line, EM2 and Exo1^-/- cell lines complement each other. (C) MSI in VCMsh2^LoxP/LoxP mice (for MSI in C57B1/6J mice see 19). (D) Western blot analysis of EM2 MEF cell lines. (Δ12), MSH2 is absent from an EM2 cell line and shows reduced amounts of its complex partner, Msh6. WT, wildtype mouse embryonic fibroblast cell line. (E) IHC on VCMsh2^LoxP/LoxP small intestine (top) compared to wildtype intestine (bottom). (F) RT-PCR using Msh2 primers with GI tissues 1) wildtype, 2) VCMsh2^LoxP/LoxP.

Figure 3

VCMsh2^LoxP/LoxP mice have decreased median survival. (A) An intestinal adenocarcinoma from a VCMsh2^LoxP/LoxP mouse. (B) Survival curves: wildtype, (green); VCMsh2^LoxP/LoxP, (red); ECMsh2^LoxP/LoxP, (blue, solid); Msh2^null (black, dashed). (C) An intestinal adenocarcinoma from a VCMsh2^LoxP/LoxP mouse stained with rabbit anti mouse E cadherin -24E10 (Cell Signaling Technology, Danvers, MA). The black square on the lower right shows tumor invasion of the muscularis and is enlarged in panel D). The smaller square on the lower right is enlarged in panel E) and shows multiple mitotic figures indicated by black arrows. F) IHC using antibody to Apc on a VCMsh2^LoxP/LoxP intestinal polyp (top), specific staining of Apc in macrophages of the intestinal epithelium (lower left), wildtype control (lower right).

Figure 4

Tumor size measurements by caliper and tumor number count, from the intestines of allelic phase mutants. (A) The average number of tumors between VCMsh2^LoxP/null and VCMsh2^LoxP/G674D mice varied significantly, as did the size (B). (C) Intestinal tumors for chemotherapy are visualized and measured by MRI. A six month old VCMsh2^LoxP mouse testing positive for occult blood, was subjected to MRI, successfully revealing one tumor. Two weeks later a second tumor was detected during a second MRI at the original location.

Figure 5

Intestinal tumors from VCMsh2^LoxP/null and VCMsh2^LoxP/G674D mice, respond to chemotherapy. Tumors are measured in terms of relativity of MRI measurements. The tumor size at day 0 is 1, and the relative tumor growth or retardation is scored on the basis of percentage. Red lines indicate growth, green lines indicate retardation. The number of tumors for each treatment is as follows. VCMsh2^LoxP/null: PBS-7, cisplatin-8, FOLFOX-11. For VCMsh2^LoxP/G674D: PBS-7, cisplatin-9, FOLFOX-11.

Figure 6

Apoptosis measured in intestinal mucosa of VCMsh2^LoxP/null and VCMsh2^LoxP/G674D mice, after 18 hours of FOLFOX treatment. (A) TUNEL staining (green fluorescence) in the intestinal mucosa of a. Msh2^LoxP/LoxP, b. Msh2^null, c. VCMsh2^LoxP/null, d. VCMsh2^LoxP/G674D mice with graphic analysis of the number of positive apoptotic cells per fifty crypts. (B) Apoptosis measured in the spleen of VCMsh2^LoxP/null and VCMsh2^LoxP/G674D mice, after 18 hours of FOLFOX treatment. A. TUNEL staining (green fluorescence) in the intestinal mucosa of a. Msh2^LoxP/LoxP, b. Msh2^null, c. VCMsh2^LoxP/null, d. VCMsh2^LoxP/G674D mice with graphic analysis of the number of positive apoptotic cells per fifty crypts.