1. To generate knockout mice of a gene of interest you classically modify the locus of interest in ES cells by homologous recombination, and in a second step then use the ES cells to generate mice with the modification. Why is it not possible to directly use zygotes for the modification?
Because of the uptake efficiency when the locus of interest is tranferred you try it with millions of ES cells and it will works in some (statistics) but you only have a limited number of zygotes -> probability that the goi is taken up properly is too low
Now with CRISPR/CAS9 the efficiency is much better and you can directly do the modification in the zygote
2. Why do you have to inject the modified ES cells into a blastocyst and then transfer this injected blastocyst to a foster mouse to generate mice from the ES cells? Wouldn't it be much easier to directly transfer ES cells to the uterus of a foster mother?
ES cells alone cannot generate extraembryonic structures they are pluripotent but not omnipotent
3. In the targeting construct for beta-Catenin in slide 5, the two homology arms are very different in length, one is quite short, the other much longer. What is the advantage of a short and long homology arm instead of two arms of roughly equal size?
Besprechen!
-> short arm needed for better PCR-Priming: with a short Primer the generation times are much shorter
-> a long arm on the other hand secures that the homology is very specific
-> allows targeting construct to be introduced in correct orientation
4. Why is a good idea to include a negative selection cassette in your targeting costruct?
Because random integration is a quite frequent event -> with the negative selection marker you select cells that performed homologous recombination at the right locus
prevent random integration with its own promotor
5. In order to express a reporter gene in the expression domain of a gene x you can either knock in the reporter into gene x, or you can generate a transgenic line using the regulatory region of gene x to control expression of the transgene. What are the main differences between these two strategies?
Transgenic Strategy: WT functions and epigenetic mechanisms still intact
Knock In Strategy: Loss of the WT function (possible but not necessarily) and potential epigenetics, many mice needed because you dont know where you integrated the gene x (you can target your knock-in via homology regions, transgenes are integrated in random regions which can affect the endogenous function as well as the expression rate and intensity of the reporter)
6. Look at the list of modified loxP and FT site in silde 24. What is the consequence of modifying the spacer sequence of a site, what effects do modifications of the inverted repeats have?
modification of inverted repeats:
right element or left element mutants, enable stable inversion, insertion, excision. after recombination: one loxP site will be completely mutated and does not bind recombinase anymore which makes the recombination irreversible
modification of spacer sequence:
allows recombination mediated cassette exchange?
—> enhances recombination efficiency
7. Look at slide 29: For generating a knock-out of FGF8 females homozygous for the floxed allel were crossed to males carrying Cre and a null allel of FGF8
a) What is the advantage using a male carrying Cre and a null allel of FGF8 instead of a floxed FGF8 allel?
b) Why don't you use a male carrying Cre that is homozygous for a floxed allel of FGF8?
c) Why don't you do the cross the other way around using males homozygous for the floxed allel females carrying Cre and a null allel of FGF8?
!Besprechen ?
a) better recombination efficiancy CRE only needs to recombine one allele
—> if Cre would have to combine both alleles, levels of Cre might be too low and therefore result in incomplete recombination
b) a homozygous individual would die -> not possible to cross
c) when you create the genetic engineered mice you have to check genotype (taking bit of tissue) if the male is correct you can always use it to impregnate more females, the females you can only use once if you need the embryos -> thats why you have the females homozygous so you are sure they always the same and you have the males with the heterozygous construct and 1/4 of the offspring will have the right combination
8. What are important factors that you have to think about, when you design a knock out strategy for a gene?
null allel should not produce functional protein !
cassette with reporter and markers (positive and negative) but potentially removeable
always prepare for later modifications -> integrate more recombinase sites
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