BCH5413 COMPREHENSIVE EXAM 2026
QUESTIONS WITH SOLUTIONS GRADED A+
◉ When determining if an additional copy of a gene added to a mouse
genome using PCR, how would you be able to distinguish between the
endogenous gene and the transgene? Answer: For PCR, I use two
primers, one inside the transgene and one outside the transgene, so I can
distinguish the endogenous gene from the transgene. Since only the
transgene can bind with these two primers and be amplified.
◉ When determining if an additional copy of a gene added to a mouse
genome using a Southern blot, how would you be able to distinguish
between the endogenous gene and the transgene? Answer: Like PCR, I
will use one probe to target the gene and another probe to target the
vector in Southern Blot. When there is only one band with the size of the
gene, then it is an endogenous gene. If there are two bands, one is with
the size of the gene, another is the size of the vector, and it is the
transgene.
◉ What would be the purpose of creating a reporter gene as a transgene?
What types of experimental questions might you be able to ask and
answer? Answer: Reporter genes are used to investigate the expression
of other genes whose expression we need to measure in a transgenic
animal. Reporter genes "report" whether the gene of interest is expressed
or not, or the location of gene expression.
,In the lecture, the example of the HKβ subunit gene is given, in which
using a reporter gene, scientists want to investigate the location of
expression of the HKβ subunit gene in the tissues of a transgenic mouse.
LacZ gene is used as a reporter and the HKβ control region is placed in
front of the LacZ gene in a plasmid vector. Now the tissues that
expressed the HKβ subunit gene also expressed the reporter LacZ gene
and it became easier for the scientists to locate the expression of the
HKβ subunit gene because of the blue color of the tissues, expressed by
the LacZ gene whose product is β-galactosidase.
Other reporter genes mentioned in the textbook include the luciferase
gene from firefly and the bacterial gene cat that encodes
chloramphenicol acetyl transferase enzyme (CAT).
◉ When creating a knockout animal, why do you need to engineer ES
cells? Answer: **Able to perform manipulation of the genome but the
cells still grow into a whole organism. ES cells give rise to all body
tissue types. iPS cells: take cells that are differentiated and cause them
too undifferentiated.
**ES cells = pluripotent, used to knock out genes of interest.
◉ When creating knockout ES cells, why do you need to isolate cells
that are sensitive to G418 AND ganciclovir? Answer: To tell you where
your transgene is being expressed!
,G418 neomycin analog kills cell without neomycin-resistance gene
(cells with no integration)
Gangcyclovir kills cells containing thymidine kinase gene (cells with
non-specific recombination)
Cells that grow on medium will have homologous recombination
between the plasmid and WT gene
◉ When creating a knockout mouse, the chimeric animal is mated with
one that is black and normal for the gene of interest. From this cross,
three possible offspring can be produced. Explain the genetics of each of
the three and what you would do next with the desired offspring.
Answer: possible progeny genomes include: A/X+ (brown, no gene of
interest), A/X- (brown, gene of interest), a/X+ (black, no gene of
interest) in which A/X- is the most desirable genome.
I would then mate two heterozygous brown mice (X+X-) containing the
gene of interest to produce a homozygous brown knockout mouse (X-X-
) and screen by southern blot.
◉ Where in the genome are loxP sites located in a Cre/Lox model?
a. Adjacent to the Cre locus expressed in a desired cell type
b. On either side of the gene that is to be knocked out
c. Random sites throughout the genome Answer: b. On either side of the
gene that is to be knocked out
, ◉ In which organism was the CRISPR system discovered? Answer:
System of bacterial natural immunity. From studying natural immunity
in bacteria.
◉ In the immunization phase, what happens to the foreign viral DNA?
Answer: Phase 1(immunization phase): how the bacterial cell makes
itself immune
Phase 2 (immunity phase): how it uses that to protect itself from a
second infection.
The foreing viral DNA is cleaved into smaller pieces called spacers. The
spacers are incorporated into the CRISPR locus; part of that region of
the chromosome has the spacers in it. The small pieces of viral DNA are
separated from each other by black boxes called repeats.
◉ In the immunity phase, what RNAs are present in the Cas9 complex?
And what is their role? Answer: pre-CRISPR RNA-contains the spacers
and repeats
RNase III-cleave the RNase between the spacer and the spacer and
repeat, cleaves between these spacers and repeats from the rest of the
spacers and repeats
tracrRNA-tracer binds to the repeat, which makes a recognition site for
the RNase III
◉ What happens in a new infection from a previously recognized viral
DNA? Answer: The second time the bacteria encounter the same viral
RNA, it will go into the cell and it will complementary base pair with
QUESTIONS WITH SOLUTIONS GRADED A+
◉ When determining if an additional copy of a gene added to a mouse
genome using PCR, how would you be able to distinguish between the
endogenous gene and the transgene? Answer: For PCR, I use two
primers, one inside the transgene and one outside the transgene, so I can
distinguish the endogenous gene from the transgene. Since only the
transgene can bind with these two primers and be amplified.
◉ When determining if an additional copy of a gene added to a mouse
genome using a Southern blot, how would you be able to distinguish
between the endogenous gene and the transgene? Answer: Like PCR, I
will use one probe to target the gene and another probe to target the
vector in Southern Blot. When there is only one band with the size of the
gene, then it is an endogenous gene. If there are two bands, one is with
the size of the gene, another is the size of the vector, and it is the
transgene.
◉ What would be the purpose of creating a reporter gene as a transgene?
What types of experimental questions might you be able to ask and
answer? Answer: Reporter genes are used to investigate the expression
of other genes whose expression we need to measure in a transgenic
animal. Reporter genes "report" whether the gene of interest is expressed
or not, or the location of gene expression.
,In the lecture, the example of the HKβ subunit gene is given, in which
using a reporter gene, scientists want to investigate the location of
expression of the HKβ subunit gene in the tissues of a transgenic mouse.
LacZ gene is used as a reporter and the HKβ control region is placed in
front of the LacZ gene in a plasmid vector. Now the tissues that
expressed the HKβ subunit gene also expressed the reporter LacZ gene
and it became easier for the scientists to locate the expression of the
HKβ subunit gene because of the blue color of the tissues, expressed by
the LacZ gene whose product is β-galactosidase.
Other reporter genes mentioned in the textbook include the luciferase
gene from firefly and the bacterial gene cat that encodes
chloramphenicol acetyl transferase enzyme (CAT).
◉ When creating a knockout animal, why do you need to engineer ES
cells? Answer: **Able to perform manipulation of the genome but the
cells still grow into a whole organism. ES cells give rise to all body
tissue types. iPS cells: take cells that are differentiated and cause them
too undifferentiated.
**ES cells = pluripotent, used to knock out genes of interest.
◉ When creating knockout ES cells, why do you need to isolate cells
that are sensitive to G418 AND ganciclovir? Answer: To tell you where
your transgene is being expressed!
,G418 neomycin analog kills cell without neomycin-resistance gene
(cells with no integration)
Gangcyclovir kills cells containing thymidine kinase gene (cells with
non-specific recombination)
Cells that grow on medium will have homologous recombination
between the plasmid and WT gene
◉ When creating a knockout mouse, the chimeric animal is mated with
one that is black and normal for the gene of interest. From this cross,
three possible offspring can be produced. Explain the genetics of each of
the three and what you would do next with the desired offspring.
Answer: possible progeny genomes include: A/X+ (brown, no gene of
interest), A/X- (brown, gene of interest), a/X+ (black, no gene of
interest) in which A/X- is the most desirable genome.
I would then mate two heterozygous brown mice (X+X-) containing the
gene of interest to produce a homozygous brown knockout mouse (X-X-
) and screen by southern blot.
◉ Where in the genome are loxP sites located in a Cre/Lox model?
a. Adjacent to the Cre locus expressed in a desired cell type
b. On either side of the gene that is to be knocked out
c. Random sites throughout the genome Answer: b. On either side of the
gene that is to be knocked out
, ◉ In which organism was the CRISPR system discovered? Answer:
System of bacterial natural immunity. From studying natural immunity
in bacteria.
◉ In the immunization phase, what happens to the foreign viral DNA?
Answer: Phase 1(immunization phase): how the bacterial cell makes
itself immune
Phase 2 (immunity phase): how it uses that to protect itself from a
second infection.
The foreing viral DNA is cleaved into smaller pieces called spacers. The
spacers are incorporated into the CRISPR locus; part of that region of
the chromosome has the spacers in it. The small pieces of viral DNA are
separated from each other by black boxes called repeats.
◉ In the immunity phase, what RNAs are present in the Cas9 complex?
And what is their role? Answer: pre-CRISPR RNA-contains the spacers
and repeats
RNase III-cleave the RNase between the spacer and the spacer and
repeat, cleaves between these spacers and repeats from the rest of the
spacers and repeats
tracrRNA-tracer binds to the repeat, which makes a recognition site for
the RNase III
◉ What happens in a new infection from a previously recognized viral
DNA? Answer: The second time the bacteria encounter the same viral
RNA, it will go into the cell and it will complementary base pair with
