BCH5413 Exam 2 UPDATED ACTUAL
Exam Questions and CORRECT Answers
What is the meaning of the term transgenic? - CORRECT ANSWER - A transgenic
organism is one whose genome has been altered by the random introduction of a foreign piece of
DNA. Researchers often compare transgenic organisms to wild type or control organisms to
determine the effect the transgene has. The amount of DNA inserted can vary from 1-200 base
pairs
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? - CORRECT
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? -
CORRECT 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? - CORRECT 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? - CORRECT
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? - CORRECT 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.
- CORRECT 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 - CORRECT ANSWER - b. On either side of the
gene that is to be knocked out
In which organism was the CRISPR system discovered? - CORRECT ANSWER - System
of bacterial natural immunity. From studying natural immunity in bacteria.
In the immunization phase, what happens to the foreign viral DNA? - CORRECT
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? -
CORRECT 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? - CORRECT
ANSWER - The second time the bacteria encounter the same viral RNA, it will go into the
cell and it will complementary base pair with the spacer because it was made from the same viral
DNA. The viral DNA will then get cleaved by the CRISPRS
What is the importance of PAM sequence in CRISPR Cas9 technology? - CORRECT
ANSWER - THE PAM sequence is present in the invading viral DNA, and it is not present
as part of the spacer. So the cell or the cas9 complex recognizes that the viral DNA is non-self
and it will get cleaved.
What are the 2 possible repair mechanisms of a double strand break? Which one of the methods
would you prefer if you wish to make a three nucleotide change in your target gene using
CRISPR Cas9? - CORRECT ANSWER - The first mechanism is to leave the double strand
break alone and in some number of cells you would get non-homologous end joining. During this
Exam Questions and CORRECT Answers
What is the meaning of the term transgenic? - CORRECT ANSWER - A transgenic
organism is one whose genome has been altered by the random introduction of a foreign piece of
DNA. Researchers often compare transgenic organisms to wild type or control organisms to
determine the effect the transgene has. The amount of DNA inserted can vary from 1-200 base
pairs
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? - CORRECT
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? -
CORRECT 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? - CORRECT 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? - CORRECT
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? - CORRECT 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.
- CORRECT 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 - CORRECT ANSWER - b. On either side of the
gene that is to be knocked out
In which organism was the CRISPR system discovered? - CORRECT ANSWER - System
of bacterial natural immunity. From studying natural immunity in bacteria.
In the immunization phase, what happens to the foreign viral DNA? - CORRECT
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? -
CORRECT 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? - CORRECT
ANSWER - The second time the bacteria encounter the same viral RNA, it will go into the
cell and it will complementary base pair with the spacer because it was made from the same viral
DNA. The viral DNA will then get cleaved by the CRISPRS
What is the importance of PAM sequence in CRISPR Cas9 technology? - CORRECT
ANSWER - THE PAM sequence is present in the invading viral DNA, and it is not present
as part of the spacer. So the cell or the cas9 complex recognizes that the viral DNA is non-self
and it will get cleaved.
What are the 2 possible repair mechanisms of a double strand break? Which one of the methods
would you prefer if you wish to make a three nucleotide change in your target gene using
CRISPR Cas9? - CORRECT ANSWER - The first mechanism is to leave the double strand
break alone and in some number of cells you would get non-homologous end joining. During this
