Exam #2 Review

Question 1

If a third bcd target gene contains only bcd
binding sites with lower affinity than those in
otd, which prediction is true for its
expression domain?

Answer 1

It is smaller than otd

Question 2

Eggs laid by a female fly with three copies
of Bcd develop into embryos with

Answer 2

Bigger heads

Question 3

Which of the following is NOT true
about a reverse transposon?

Answer 3

It contains a gene for viral coat

Question 4

Which of the following is NOT an
advantage of gene-targeting?

Answer 4

It allows you to discover new genes involved
in a specific developmental process

Question 5

Why is HSV-tk part of a gene-targeting
vector?

Answer 5

It provides a mechanism of negative selection because it converts Gancyclovir into a toxin that kills the cells.

Cells with random insertion of the vector contain this
gene and will be killed by the drug

Cells with targeted insertion of the vector will not
contain this gene and will survive the drug selection

Question 6

Which of the following is NOT correct?

Answer 6

The zinc-finger nuclease contains the DNA-
binding domain of FokI

Question 7

What is TALEN, and how does it work?

Answer 7

• A TALEN is a fusion protein between multiple
  TALE domains and an endonuclease domain.
• Each TALE domain recognizes a specific
  nucleotide through two amino acids near its
  center
• The TALE domains provide the DNA sequence
  specificity and the nuclease domain make the
  double stranded breaks.

Question 8

Which of the following is NOT true
about CRISPR/Cas9 system?

Answer 8

The cell is always able to fully repair the double-
stranded breaks generated by the CRISPR/Cas9
complex

Question 9

Which of the following is NOT true
about in ovo electroporation?

Answer 9

The genetic changes generated by this method
can be easily passed onto the next generation.

Question 10

If a DNA contains two loxP sites facing each
other, what will happen when Cre mediates the
recombination between these loxP sites?

Answer 10

The fragment between them will be inverted

Question 11

What is NOT true about base editing?

Answer 11

Base editing leads to high level of non-
homologous end-joining

Question 12

Which of the following is NOT part
of the MCR cassette?

Answer 12

A gRNA targeting Cas9

Question 13

Which of the following is NOT
true about CATCHA?

Answer 13

The CATCHA allele behaves as a recessive
allele in a Cas9-expressing population

Question 14

What is the biggest advantage of
enhancer trap?

Answer 14

It can trap genes at higher frequency than
other trapping methods

Question 15

Why did b-geo replace b-gal as the more
popular vector type for gene-trapping?

Answer 15

It increases the frequency of gene-trapping
among G418 resistant cells

Question 16

The figure below shows a double transgenic mouse
embryo. You inject the pregnant mouse with Tamoxifen at embryonic day 10 (E10). Assume that CMV promoter is active throughout development and Tamoxifen is active for two days in the animal, is the transgene expressed at E18? Why?

Answer 16

Yes!
Once the Stop sequence is removed by
the recombination between the two loxP
sites, the transgene remains on.

*ERT is a mutated Estrogen receptor that can enter
the nucleus upon binding with Tamoxifen

Question 17

The figure below shows a double transgenic mouse
embryo. You inject the pregnant mouse with Doxycycline at embryonic day 10 (E10). Assume that CMV promoter is active throughout development and Doxycycline is active for two days in the animal, is the transgene expressed at E18? Why?

Answer 17

No!
Doxycyline binding to rtTA can transiently
activate transgene expression. Once the drug
is degraded (around E12), the transgene will
be turned off.

Question 18

Which of the following is NOT true
about a DNA transposon?

Answer 18

It moves around the genome in a
“copy and paste” fashion.

Question 19

When using transposons as an insertional
mutagenesis tool, why is removing the
transposase important after transposition has
occurred?

Answer 19

If the transposase is not removed, the transposon will continue to move around the genome, so we cannot keep a stable heritable mutation to study gene function. Therefore, transposase has to be removed before further analysis of the mutant phenotype.

Question 20

What is not included in an
enhancer trap vector?

Answer 20

Enhancer

Question 21

Which trap method has the highest
trapping frequency?

Answer 21

Enhancer trap

Question 22

Which of the following is NOT correct
about the ES cells?

Answer 22

They are easy to manipulate in culture

Question 23

Place the following events in order. (1) The
Holliday Junction is resolved leading to the
separation of the two homologous DNA. (2)
DNA double-strand break occurs. (3) The gap is
filled by DNA polymerase. (4) The broken DNA
strands pair with their homologous DNA
strands.

Answer 23

2-4-3-1

Question 24

What does tk do?

Answer 24

It converts Gancyclovir into a toxin that
kills the cells

Question 25

which of the following statements is
correct?

Answer 25

b-Gal trap vectors lead to more G418 resistant cells than b-Geo ones

Question 26

Which of the following is NOT
part of a polyA trap vector?

Answer 26

polyA signal

Question 27

Which of the following statements about the
second generation promoter trap is NOT true?

Answer 27

Its insertion in an intron allows reporter expression

Question 28

What’s common in all the mutagenesis
approaches we have learned so far?

Answer 28

Every cell in the mutant animals carry
the same mutation

Question 29

After in ovo electroporation, only one side of
the brain expresses the transgene. This is
because

Answer 29

The DNA is negatively charged and only migrates to
one side of the brain

Question 30

To mutate gene X specifically in the eyes,
which of the following do you need?

Answer 30

• A mouse with two loxP sites inserted in gene X
• A mouse that express Cre in the eyes

Question 31

Why is the efficiency of gene-
targeting very low?

Answer 31

DNA double stranded break (DSB) at a specific site
is a rare event

Question 32

Which of the following is NOT
true about ZFN?

Answer 32

The two ZFNs in a dimer have to target the same
DNA sequence

Question 33

Which of these statements about
Cas9 is FALSE?

Answer 33

It recognizes a 5’ TTN PAM

Question 34

Which of the following is true?

Answer 34

In the tet-ON system, the tetO-driven transgene is
only activated when Doxycyclin is present

Question 35

What is Cre/loxP Used For?

Answer 35

 It is used to control gene activity
spatiotemporally (location and time).

 Can be used to target almost any tissue of the
mouse to help us study gene function.

 Cre/loxP can be used to target sophisticated
systems that would be hard to study otherwise.
Studying these systems is often effective in
research of pathogenesis of human diseases.

Question 36

If you would like to express a transgene
between embryonic day 9 and 10, which
system are you going to use?

Answer 36

Tet-ON, administer Doxycycline at day 9

Question 37

What is the major difference between
BE3 and BE2?

Question 38

What is a morphogen?

Answer 38

Diffusible substance that carries
information relating, for example: to
position in the embryo, and thus
determines the differentiation that cells
perceiving this information will undergo.

Question 39

A morphogen has to…

Answer 39

• Form a simple concentration gradient (linear
  or exponential)
• Use its concentration as the main
  mechanism to influence cell behavior
• Directly regulate all cells without having to
  get help from secondary signaling

Question 40

Which of the following could be a
morphogen?

Answer 40

Signal D: induce various digits in the chicken limb based on their distance to the signal producing cells.

Question 41

If a protease that specifically targets Bcd for
degradation is blocked by a drug, what would you
expect to see in the resulting fly?

Answer 41

It will have a smaller abdomen