Genetically modified mice Flashcards
What defines a transgenic animal?
- A synthetic gene or a modification of an existing gene (by addition of external DNA), is introduced during early development
- The genetic change is incorporated into somatic and germ cells and is inherited according to Mendelian laws
What can transgenic animals be used for?
- basic research
- disease models
- bioreactors
What does pronuclear injection lead to?
- over-expression
- misexpression
- expression of mutated proteins
How does pronucelar injection work?
- DNA microinjected into fertilised mouse egg
- DNA integrates randomly into a chromosome (several copies, but only ONE integration site)
- The transgene contains its own regulatory region governing gene expression
- Chromosomal regulatory regions or DNA strucutre can disturbed the planned expression pattern
Describe the workflow of pronuclear injection
- Superovulated egg donor (fertilized eggs)
- DNA injection 1-5ng/mL 1 pl/cell
- Implantation in pseudopregnant female
- pups born 3 weeks later
- tail biopsy
- DNA analysis: detection of transgene
What are founder mice?
The pups following pronuclear injection that carry the transgene (20-30% of all pups). Each of these funders gives rise to a separate transgenic mouse line (crossed with WT, 50% of offspring are transgenic)
What is blastocyst injection / how does it work?
- change a specific gene in the chromosomal DNA (not random)
- very unusual event - direct microinjection of DNA does not work efficiently
- genetic modification is done in embryonic stem cells
- genetically modified stem cells are injected into early embryo (blastocyst)
Which kind of transgenic animals can be produced with blastocyst injection?
- knock-out mice
- deletion of a specific gene in a whole organism
- knock-in mice
- exchange one gene for another
- WT changed to a totally different gene
- WT changed to a mutated gene
- exchange one gene for another
- conditional knock-out mouse
- inactiviation of a specific gene in a specific tissue and or at a specific time point
How to prepare for blastocyst injection
- design and construct DNA targeting vector (containing antibiotic resistance gene)
- transfect ECS cells with targeting vector –> homologous recombination needed!
- grow cells with antibiotics, only cells that have integrated targeting vector survive
- pick individual colonies
- analyse the correct ESCs
- ESCs are expanded, injected to blastocysts and transferred to pseudopregnant female
What is a chimera?
- A mouse that carries the ESCs in addition to its own genes
- When germline transmission, it can transmit modification to the next generation
Workflow of blastocyst injection
- isolate BC
- culture ES cells
- gene targeting
- genetically modified ESCs
- inject into blastocyst
- reimplant into foster mother
- get chimeras
- cross two heterozygous chimera
- 25% of offspring will be homozygous for KO/will be KO
- mate these homozygous mice if viable and fertile
Solutions to the problem with the background strain
- breed your mutant strain with the desired background strain (about 10 generations)
- make the mutation directly in the desired strain
- pronuclear injection:
- different strains are more or less responsive to superovulation.
- eggs from various mouse strains differ in pronuclear size, survival rate after injection etc
- blastocyst injection
- Only ESCs from very few strains available
- pronuclear injection:
What to do when the transgene is not expressed when/where expected
(surrounding DNA sequences disturb expression)
- use transgene with very long control regions
- insert DNA in permissive chromosomal region where expression is not disturbed
Name three new tools for genome engineering
- zinc finger nucleases (ZFNs)
- transcription activator-like effector nucleases (TALENs)
- CRISPR/Cas (RNA guided cleavage)
Based on sequence specific cleavage of genomic DNA, requires NHEJ
