Genetically modified mice

• 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

• basic research • disease models • bioreactors

• over-expression • misexpression • expression of mutated proteins

• 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

• 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

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)

• 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)

• 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 • conditional knock-out mouse • inactiviation of a specific gene in a specific tissue and or at a specific time point

• 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

• A mouse that carries the ESCs in addition to its own genes • When germline transmission, it can transmit modification to the next generation

• 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

• 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

• use transgene with very long control regions • insert DNA in permissive chromosomal region where expression is not disturbed

• 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