Maximizing mouse cancer models: Notes Laboratory mice ( Mus musculus) are ideal for cancer investigations Often

ncer investigations Often use xenograft tumors derived from tumor tissue, aka cell lines or animal culture Cell autonomous- a trait only in cells with cancer mutation Non-cell autonomous a trait in cells without mutation Xenografts are easy and fast, but differences with actual tumors in live specimens However, original tumor architecture is lost and observation of improper tumor microenvironments Autochthonous tumor- tumors derived from normal cells, not implanted Environmentally induced tumors allow for identification of oncogenes, but cannot develop all types of tumors and do not precisely tumors of a certain power. Genetically engineered models are the most sophisticated. Transgenic GEM are mice that express oncogenes or dominant-negative tumor-suppresor genes in a non-physiological manner owing to ectopic promoter and enhancer elements. Ectopic- meaning displaced; implying promoters and enhancers are unnatural in that particular cell, allowing for the expression of those cancer genes Produced by injection of fertilized eggs or gene targeting in embryonic stem cells Sometimes, the transgene injected into the DNA may not necessarily be expressed, requires the proper promoters and transcription factors Transgene put into euchromatin, chromatin that is expressed, may circumvent problem. Still needs promoters so expression levels are unpredictable .reversibly control target-gene expression with exogenous ligands. Use the tetracycline operon to promote or inhibit transcription Oncogene addition: tumors arise by becoming dependent on oncogene expression overexpression of oncogene may cause senescence or apoptosis Estrogen receptor fusion proteins may be mutated, misfolded to cause oncogene function, drugs to mediate their effects Vertical transmission of transgenic GEM Viruses used to deliver cDNA into tissues Endogenous GEM are mutant mice that lose expression of TSGs or oxpress dominantnegative TSGs.. gene knockout Cause a loss of heterozygosity or haploinsufficiency to determine tumorigenic potential of mutant alleles There are two basic technical approaches to produce genetically modified mice. The first involves pronuclear injection into a single cell of the mouse embryo, where it will randomly integrate into the mouse genome.[5] This method creates a transgenic mouse and is used to insert new genetic information into the mouse genome or to over-express endogenous genes. The second approach involves modifying embryonic stem cells with a DNA construct containing DNA sequenceshomologous to the target gene. Embyonic stem cells that recombine with the genomic DNA are selected for and they are then injected into the miceblastocysts.[6] This method is used to manipulate a single gene, in most cases "knocking out" the target gene, although more subtle genetic manipulation can occur (e.g. only changing single nucleotides). Mutations of stem cells may cause undesirable-effects outside of the tissue of interest Study familial cancer diseases Conditional models: GEM that rely on site-specific recombinase systems to engender gene expression in a spatially or temporally restricted manner

Use of the Cre-Lox bacteriophage Cre recombinase finds inverted repeat DNA elements, and deletes them, in the exons Forms unstable gene product Use the Lox Stop Lox cassette between the promoter and coding exon of oncogenic allele. Excision of cassette causes oncogenic expression Can find deletions, translocations, and inversions that cause oncogenic expression Latent allelles- stochastic recombination event is required to activate expression of oncogenic allele, does not need tissue specific mouse Inadequate mRNA expression may influence other genes Problems: genetics of human cancer must guide generation of mouse models Mutation of pathway rather than specific gene is not as accurate Putting Lox Stop Lox cassette between promoter and coding sequence will make mice functionally heterozygous before expression of Cre. Non-cell autonomous contribution to carcinogenesis Use minigene cassettes that express wild-type allele before recombination and mutant after Cre activation Mutant protein is not equal to absence of wild-type protein Neomorphic allele- mutational conveying new activity not present in wild type Hypomorphic allele- mutation conveying decreased activity Limit the cancer-initiating population because in reality, tumorigenesis starts with only 1 cell. Restrict expression of active SSR The latent allele is the most accurate model but no method to control tissue specificity Tumor initiating stem cells are small part of total tumor cells Carcinogenesis starts with 5 or 6 mutant alleles, study the synergistic effects of multiple mutations Humanize mice to model biology of humans o Insert human genes into mouse genome; knockin o Recombinase-mediated genomic replacement: replace mouse genome with human genome. o Replace murine telomeres with human telomeres o Metabolism of drugs: difference in xenobiotic receptors and cytochrome P450. o Xenobiotic receptors: enzymes that sense the drug and initiate response o Cytochrome P450: enzymes that detoxify and modify drugs o Add in components of human immune system into the murine immune system. Knock in human loci for T-cell and B-cell receptors. And MHCs Importance of the microenvironment Detect the disease by indication of lethargy, use MRI and PET.