Study questions Flashcards
What is the main difference between a random integration transgenic (RIT) mouse and a knock-in
(KI) mouse?
. RIT mice carry the transgene in a random location in the genome, whereas KI mice carry a
transgene in a specific genomic location chosen by the developer.
What are the advantages of a random integration transgenic mouse?
RITs are easy to make and can carry multiple copies of the transgene leading to high expression
levels.
What are the disadvantages of a random integration transgenic mouse?
Because RITs integrate into random locations, it cannot be predicted ahead of time how the
neighboring sequences in the integration site will influence transgene expression. Furthermore, since
any two founder lines will have the transgene in different genomic locations it cannot be expected
that both will have the same expression patterns. There is also the opposite problem where the
transgene can influence the native locus, for example by mutating a gene or altering its
neighborhood.
A second problem is that lines with multiple copies of the transgene can be unstable, leading to a
reduced copy number in later generations, and thus a potential for different phenotypes in earlier
versus later generations.
What is a chimera? Describe very briefly how it is made.
. A chimera is made by fusing embryonic stem cells (ESCs) with an early stage mouse embryo. This is
typically done by injecting ESCs into the blastocoel of 3.5 day old embryos (blastocysts). These
injected embryos are then transferred to the uterus of a foster mother where they develop like
ordinary mice. The result is that ESCs are embedded in a living mouse called a chimera, and if the
ESCs contribute to the germ cells of the chimera, mutations engineered into the ESCs while grown in
culture can be carried by the progeny of the chimera.
What should be done before performing an injection of ES cells into a host blastocyst?
a) Testing for the presence of mycoplasma
b) Karyotyping
c) Verification of the correct recombination event (by PCR or southern blotting)
d) All of the above
D
Out of the four strategies, what is likely to be the most efficient in obtaining correctly recombined
clones (e.g. highest fraction of resistant colonies)?
a) Using a positive and negative marker genes on a targeting construct, e.g. neomycin
resistance gene (neo) and thymidine kinase gene(TK)
b) Using only a positive marker gene on a targeting construct, e.g. neomycin resistance gene
(neo)
c) Using only a negative marker gene on a targeting construct, e.g. thymidine kinase gene(TK)
d) Using a promotorless marker gene on a targeting construct and targeting it to a region
containing a strong promoter
2
e) Using a promotorless marker gene embedded in the genomic region of interest and
providing a promoter for it via recombination with targeting construct carrying a promoter
E
Where on a targeting construct should a negative selection marker gene be placed:
a) Between the homology arms
b) Outside of the homology arms
c) A negative marker gene is never placed on a targeting construct, as it would prevent efficient
electroporation
B
The proportion of positively recombined clones amongst the picked colonies can be improved by:
a) inducing a double-strand break (DSB) in close proximity of the recombination site
b) the CRIPSR/Cas9 system
c) using a positive and negative selection markers on targeting vectors
d) targeting a marker gene into the destination locus first, and then selecting for the loss of this
marker gene
e) increasing the concentration of a selection agent (e.g. G418) in the medium
f) all of the above
g) a, b, c, d are correct
G
With the LoxP sites in the following orientation(facing the same direction) , the neo (neomycin resistance) gene will be:
a) inverted
b) removed
c) cleaved
d) duplicated
e) cleaved and removed
B
To induce a double-strand break (DSB) in a specific DNA sequence, CRISPR/Cas9 system requires:
a) guide RNA (gRNA)
b) Cas9 nuclease
c) Cas9 nuclease and guide RNA (gRNA)
d) None of the above
C
After a blastocyst injection of ES cells with a knocked-in transgene T, a chimeric mouse was
obtained. By breeding this chimeric mouse to a wild type mouse we could expect to obtain:
a) Wild-type mice and chimeric mice (at 1:1 ratio)
b) Wild-type mice and mice heterozygous for the transgene T (at 1:1 ration)
c) 100% transgenic mice homozygous for the transgene T
d) 100% transgenic mice heterozygous for the transgene T
e) All of the above are possible
B (D but unlikely)
Which statement is false?
a) A positive selection marker can be removed from the genome after gene targeting.
b) HPRT (Hypoxanthine-guanine phosphoribosylotransferase) is a selection marker gene that
can be selected both for (positive selection) and against (negative selection).
c) Chimeras always give progeny positive for the transgene.
d) Knocked-in transgenes are much less likely to show expression pattern variances than
randomly integrated transgenes.
C
What are some unexpected things that happen with site-specific recombinases such as Cre or
Flp? (Circle all that are true)
a) In cells homozygous for a floxed (flanked by lox P sites) allele, recombination happens
between the two alleles resulting in chromosomal rearrangement.
b) In cells homozygous for a floxed allele, recombination happens for only one allele
c) Flp recombinase recombines loxP sites
d) In cells with two different floxed alleles (different genomic loci), recombination happens at
only one allele
e) In cells with two different floxed alleles (different genomic loci), recombination happens
between the different loci, causing a chromosomal translocation
f) Recombination happens in the wrong cells (e.g germ cells).
A,B,D, E, F
Leukemia inhibitory factor (LIF) and ES cell tested fetal bovine serum (FBS) are supplements
commonly used in media for mouse ES cells to maintain pluri/totipotency. How are tissue culture
plates treated in order to maintain ES cells in a totipotent state? (Hint: remember what you saw
while picking colonies)
. Plates are first treated with gelatin, then feeder cells (mouse embryonic fibroblasts) are plated
onto the gelatinized surface. Once the feeder cells are well attached ESCs can be plated. The gelatin
aids in cell attachment of both feeders and ESCs; the feeders produce signaling molecules that
promote maintenance of ESC pluripotency, as well as providing attachment substrate for ESCs.
