• Abstract
• Table of Contents
• Figures
• Literature Cited

Abstract

Since the late 1980s, the tools to generate mice with deletions of tumor suppressors have made it possible to study such deletions in the context of a whole animal. Deletion of some tumor suppressors results in viable mice while deletion of others yield embryo lethal phenotypes, cementing the concept that genes that often go awry in cancer are also of developmental importance. More sophisticated mouse models were subsequently developed to delete a gene in a specific cell type at a specific time point. Additionally, incorporation of point mutations in a specific gene as observed in human tumors has also revealed their contributions to tumorigenesis. On the other hand, some models never develop cancer unless combined with other deletions, suggesting a modifying role in tumorigenesis. This review will describe the technical aspects of generating these mice and provide examples of the outcomes obtained from alterations of different tumor suppressors. Curr. Protoc. Mouse Biol. 2:9?24 © 2012 by John Wiley & Sons, Inc.

Keywords: knockout; knock?in; translocation; transgenic mouse; phenotype analysis

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Table of Contents

• Introduction
• Gene Deletion via Knockout Targeting Strategies
• Characterizing Tumor Models
• Generation of Mice with Deletions of Tumor Suppressors
• Generating Conditional Loss‐of‐Function Alleles
• Conditional Loss‐of‐Function Alleles for Tumor Suppressors Bypass Lethality and Expand Tumor Phenotypes
• Generating Knock‐In Alleles
• Translocations
• Transgenic Mice
• Allele Preservation
• Literature Cited
• Figures

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GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Figures

• Figure 1. A p 53‐null allele. This diagram represents a p 53‐null allele that was generated by deletion of part of intron 4 and exon 5, with insertion of neo . The neo gene does not contain a polyadenylation (poly A) site and is forced to use an endogenous poly A signal upon integration limiting the number of random insertions. MC1‐tk encodes for the thymidine kinase. FIAU: 5‐iodo‐2′fluoro‐2′deoxy‐1‐β‐D ‐arabino‐furanosyl‐uracil. Numbers above boxes denote p 53 exons.

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• Figure 2. A p 53 conditional loss‐of‐function allele. The initial step introduces three lox P sites (red ovals) and a neo‐tk gene by homologous recombination into the p 53 locus. Cre‐mediated deletion of neo and not deletion of the entire p 53 gene is possible when Cre is weakly expressed. The Trp53F2‐10 allele then contains two lox P sites, which upon recombination delete p 53 exons 2‐10 in a tissue specific manner depending on Cre expression. Numbers above the boxes denote p 53 exons.

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• Figure 3. Double‐replacement strategy to generate a point mutation in p 53. In the first recombination step, PGKneoNTRtkpA , which transcribes both neo and tk genes, replaces p 53 exons 2‐6. G418 selection is used to narrow the number of ES cell colonies examined. In the second step, a correct p 53 gene containing a single point mutation in exon 5 (*) replaces PGKneoNTRtkpA and reconstructs the locus. This targeting construct also contained an unwanted deletion of a G nucleotide at a splice acceptor site. Numbered boxes denote p 53 exons.

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• Figure 4. A knock‐in p 53 allele. To generate a p53 knock‐in allele with a single point mutation, the targeting construct contains the PGKneo cassette flanked by lox P sites (red ovals) and the point mutation in exon 5. Cre‐mediated recombination deletes the PGKneo cassette and results in the knock‐in allele with a point mutation and a single residual lox P site in intron 4. Numbers above the boxes denote p 53 exons.

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• Figure 5. Chromosomal translocations. To generate chromosomal translocations, two alleles were generated each containing half of the Hprt gene, a resistance marker neomycin (neo) or hygromycin (hygro) and a lox P site. Addition of Cre recombinase brings the IgH enhancer region upstream of the coding exons (2 and 3) for c‐Myc causing inappropriate over expression of c‐Myc . The 5′ and 3′ ends of Hprt gene also come together and allow for selection by hprt .

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