Mouse Models in Cancer Flashcards
what common mutation is seen in melanomas?
Braf or Nras
what are driver mutations?
confer an advantage to the cell in which they occur, are causally implicated in cancer development and are positively selected in tumour evolution (e.g. bcl-2)
what are passenger mutations?
are preexisting mutations that have not been selected for in tumour evolution. They were present in the cell that was the progenitor of the final clonal expansion of the cancer, are biologically neutral and do not confer an advantage
what are different methods to find out whether you are looking at a driver or a passenger mutation?
which is correlative and which is causal?
- Bioinformatics: mathematical methods of looking at mutations – correlative
- direct experimentation: determine what the gene does – causal
why make a mouse model of human cancer?
to study what?
- Understand the effects of oncogene activation or tumour suppressor loss (i.e. specific gene mutations) in normal cells within a whole organism (causality)
- determine the nature of cooperation between genetic events and how they contribute to disease (is there a sequence of events leading to the disease)
- understand the non-cell autonomous interactions (role of stroma, immune system, hormonal controls)
- to develop preclinical models to assess future therapeutics
why not just use cells in culture?
- Tissue culture conditions are permissive: Adapted for cell proliferation and protection against death
- Tissue micro-environment is altered: Cultures cells have disrupted tissue architecture (lacking somatic tissue and immune system effects)
- Culture systems do not allow for testing preclinical novel therapeutics that may target cancer supporting tissues (vasculature).
- Culture based oncogenic transformation assays can give misleading results
why the mouse and not other model systems?
- genome is sequenced and similar to humans (>80% 1:1 gene orthologs)
- relatively short generation time (9-12 weeks from birth to birth)
- genetically and biochemically malleable
- structirally similar to humans and they naturally get cancer
what are disadvantges of using a mouse model?
- expensive
- time consuming
what are the properties of a good mouse model?
- recapitulate key pathophysiological aspects of disease (same tumor types, progression, metastasis)
- employ the same mutations found in human tumors
- useful as a tool to evalute pharmacologic agents
what are the different types of mouse models?
- xenograft models
- Genetically Engineered Mouse Models (GEMMs)
- Chemical Mutagenesis Models
how do we make xenograft mouse models?
you implant foreign tumor/cancer cells into an immunocomprimised mice (e.g. FOXN1-KO leading to an unfunctional thymus and no b-cells)
how are xenografts most frequently used?
can test a drug vs solvent control to see if less tumor growth is seen
what are the pros of unsing a xenograft mouse model?
- can use human cells (test against human targets, more representative of complex genetic events seen in human cancer cells)
- highly reproducable and controllable
- quick and inexpensive
- can be used to study speciifc processes in cancer development (can study tumor viability, extravasation, and metastasis)
what are the cons of using xenograft mouse models?
- Often difficult to determine which genes are important in the process being studied (many mutations in a single cancer cell line)
- Do not allow the study of tumor / immunology interaction (mice are immuno-compromised)
- use cell lines that have been adapted to live in cell cultures (but can now do patient-derived xenograft so not so much of an issue)
- are not predictive of patient reponse to drugs
what are the two major and the one minor way to genetically engineer mouse models?
- Transgenic approach (random integration) – major
- Targeted gene approach (specific integration) – major
- Viral transduction approaches – minor
what are transgenic mice?
Mice that express a gene (or inhibitory RNA) under a specific promoter
integraton often random, most often in tandem array
what is the targeted gene approach in genetically engineering mice?
alter a specific gene as desired
(eg. knock-out, knock-in, alter promoter, include reporter etc)
what is the viral transducer approach in making genetically engineered mice?
introduce genetic information stably or transiently using virus as vector
(eg. lentivirus, adenovirus)
can we make transgenic mice specific?
yes, we cna make it tissue specific by selecting a tissue specific promoter (e.g. MMTV promoter in mammary epithelium and tyrosinase promoter in melanocytes)
what are pros of unsing transgenic mice?
- Many, many promoters can be used (tissue specific, cell type specific)
- Almost any cDNA, miRNA, shRNA can be expressed from a specific promoter
what are cons of using transgeic mice?
- Levels of expression dictated by the promoter - might not be physiological
- Random integration site - can cause problems with expression – potentially could integrate into a gene
what happens when you express c-Myc and H-ras under the MMTV promoter? what does this tell us?
both seperately: get solitary tumors
when crossed: mice get tumors all over
this does not tell us much since the levels of myc and ras are not physiological (and h-ras not expressed in breast cancer)
how can we temporally regulate transcription using transgenes?
using the “tet-off” molecule (tTA):
* instert tet transactivator under a tissue or cell specific promoter
* then, gene is only expressed in the absence of doxycyline (so cna give dox to mice to prevent expression of the gene)
* so transgene expression is temporally controlled and expressed only in region of interest
can also have reverse-tTA where dox is required for gene expression
what happens when ErbB2 is inserted at the MMTV promoter under reverse-tTA control?
when doxicycline is given, we see breast tumor formation, but we see regression of the tumor when the dox is withdrawn –tumors require sustained ErbB2 expression
but fully regressed mice then devlop tumor independent from ErbB2 espression, suggesting the presence of a population of cancer stem cells remaining after ErbB2 expression stopped.
