1. Compare the tetracycline and the tamoxifen inducible system for controlling Cre-activity. What are important differences?
Tamoxifen:
Numbers of transgenes
side effects
activation speed
Tamoxifen
2 Transgenes needed
interaction with estrogen / steroid receptors
after activation only translocation -> fast recombination
Tetracycline
3 Transgenes needed
no interactions with WT mouse physiology (but three transgenes can have impact)
transcription and translation of CRE and then recombination
2. Explain, how you can analyze what the Shh expressing cells in the early limb bud of a mouse embryo will give rise to a later stages.
How can you study what the SHH positive cells in the limb bud at a particular stage will give rise to?
-> with a tamoxifen or TET system
Shh target gene promoter in control of Cre Recombinase
transgene in Rosa26 locus with floxed stop cassette in front of lacZ encoding region
at particular stage: Cre::ER
Reporter line with Cre under control of Shh reporter
Shh promoter that drives Estrogen-Tamoxifen system at a later timepoint
3. Look at slide 48 (Combinatorial labeling of neurons). Explain panel c (Brainbow- 1.1 and Brainbow-2.1). What could the colored symbols on the right (colored ring, circle, or circle with a colored dot in the middle) indicate?
colored ring: expressed in membrane
circle: expressed everywhere
circle with a colored dot in the middle: expressed in nucleaus
4. Explain the mechanism of dual RMCE.
Dual Recombination mediated cassette exchange
knockout first strategy by EUCOMM
generation of ESCs with reporter and antibiotics resistance cassette flanked by FRT sites and exons/loci of interest floxed
expression of FLP and CRE enable incerased recombination efficiency
FLP expression first, knockout of reporter and selection cassette —> restores wildtype function
Cre expression excises goi
donor vector flanks mutated goi and new selection cassette with FRT site and loxP site
insertion of donor locus
5. Using Crispr/Cas9 to modify a gene in ES cells is much more efficient that using the traditional gene targeting approach with out Crispr/Cas9. Why is that so?
guided double strand break at right position without need for cassette system or random double strand break where its inserted
much more precise, faster and simpler
it is scaleable -> multible genes can be edited at once (different gRNAs
Efficiency of direct homology repair (or NHEJ) to introduce new DNA template
CRISPR possible in any organism (not only for organisms where ES cells are available
high targeting efficiency allows direct genome editing in zygote and not time-consuming step with ESCs necessary
—> no backcrossing needed since genome-edited offspring is not chimeric
6. What are potential problems when using the Crispr/Cas9 system?
Off target effects!
mosaicism (not all cells might be genetically modified)
CAS9 protein and the gRNA might cause immun response
the delivering system of the components
Ethical concerns
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