Mouse Transgenics Flashcards
Why generate transgenic mice?
Learn about gene function in mammals
What is a transgenic mouse?
Inserting a gene of interest (with promoter) randomly into the genome - over express a particular protein in a specific tissue/tissues
What is a knockout mouse?
Deleting all or part of a specific gene; either tissue specifically or globally
What is a knock-in mouse?
Permits generation of specific mutations in a gene of interest
What is the one cell stage important for?
Generating transgenic mice
What is the blastocyte stage important for?
Embryonic stem cell
What are the basic steps in making a transgenic mouse?
- Make transgene
- Pronuclear injection
- Mouse will give birth
- Genotype putative founders
- Breed founders to derive transgenic lines
- Test expression
What are the drawbacks of creating transgenic mice?
Position effects on the promoter of the transgene
Number of integrated transgenes is random
Integration into another gene leads to complicated phenotypes
Why use BACs?
Larger genes / gene regions
Expression levels are position independent
Better mimic of endogenous expression pattern
What is a global knockout vs conditional knockout?
Global knockout - generating a mouse which is deficient in a gene in all tissues
Conditional knockouts - tissue specifically delete a gene
What are the steps to make a knockout mouse?
- Make transgene
- Gene targeting in ES cell culture
- Selection of successfully transformed cells
- PCR-based screening
- Blastocyte injection
- Breed chimeras
Name some issues with knockout mice
Targeting vectors can take a long time to generate
Generation of knockout mice takes time
Could lead to lethality
Difficult to identify positives via PCR
Difficult to obtain high-percentage chimeras
What are the uses of the Cre/loxP system?
Tissue-specific knockout
Conditional transgene expression
Introduce subtle gene mutation (knock-in)
What needs to be considered for inducible transgenes?
Low or no activity when not induced
Large increase between induced and non-induced gene expression
Expression rapidly turns on or off
Inducer - not toxic and easily administered orally
What are the uses of Rosa26 locus?
Used to confirm Cre reporter lines via conditional expression of lacZ
Allows generation of transgenics with single insertion event at specific locus - eliminates position effects
