An Msh2 Conditional Knockout Mouse for Studying Intestinal Cancer and Testing Anticancer Agents

Department of Medicine/Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Gastroenterology (Impact Factor: 16.72). 11/2009; 138(3):993-1002.e1. DOI: 10.1053/j.gastro.2009.11.009
Source: PubMed


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.
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.
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.
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.

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Available from: Raju Kucherlapati, Oct 02, 2015
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    • "To further assess the imaging method, mice with conditional knockout (CKO) mutant alleles were generated as previously described25, 26, 27, 28 and ApcCKOp53flox/floand ApcLoxP/ LoxPMsh2null/LoxPmutant mouse models were used to evaluate the imaging potential of 64Cu-DOTA-cetuximab-F(ab′)2 in CRC. "
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    • "These differences may result in changes in chromosome stability or gene expression that are not solely due to MMR deficiency [31,32]. Matched normal cell systems, such as MMR-deficient embryonic kidney cells 293T compared to 293T cells engineered to overexpress MLH1 [33], or wild-type mouse embryonic fibroblasts (MEFs) compared to MSH2-deficient MEFs [34] may be not be ideal models to study MMR function in cancer. MEFs may be less sensitive to defects in DNA repair than cancer cells, and have been reported to exhibit a much lower mutation rate than MMR-deficient cancer cells [31,35]. "
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    ABSTRACT: The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells.
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