BCH 5413 - EXAM 2, MODULE 1 UPDATED ACTUAL
QUESTIONS AND CORRECT ANSWERS
Question:
1. What does transgenic mean?
Answer:
Refers to an organism that has been genetically altered to contain additional DNA
(from the same organism or others) in order to alter expression of specific
phenotypes to compare with wild type expression
Question:
2. When creating a transgenic mouse, how many copies are
inserted and where will they insert into the genome?
Answer:
Between 1-200 copies of the transgene can be inserted into the genome at
random.
Question:
3. Which of the following statements is true about a
transgenic mouse?
Answer:
The transgenic mouse may have multiple copies of the transgene
3 multiple choice options
Question:
4. Where is the transgene injected into the mouse?
Answer:
Into a fertilized oocyte (DNA directly injected within the male pronucleus) or
embryo (using a retrovirus). The surviving embryos are then implanted back into
the donor female
Question:
5. Once offspring are obtained from the donor mouse, how
are they determined to have the transgene(s)?
Answer:
Through the use of PCR (using transgene specific primers) followed by southern
blotting and comparing the transgenic mice genes to the wild type (endogenous)
genes. This process can confirm transgene presence as well as the copy number
of the gene.
,Question:
6. When determining transgene presence using PCR, how
can you distinguish between the wild type and
transgenes?
Answer:
Specific primers can be used - one primer will be shared between the transgene
and the endogenous gene and one will be unique to the transgene/cloning
vector (another option would be to add one unique to the endogenous gene, if
possible, for hetero/homozygous comparisons). Using these primers should
create banding that can be used to identify the presence or absence of the
transgene.
Question:
7. What is used as a control for the PCR reactions for
determining wild type or transgene presence?
Answer:
An unrelated universal mouse gene. This gene will be a good negative control for
transgene presence because it will not react with primers specific to the
transgene/vector and not produce a band to showcase lack of contamination.
Additionally, primers for this gene can be used as a control for to illustrate lack of
contamination when comparing to the wild type because it will have banding with
the universal gene but not with the transgene primers.
Question:
8. When determining transgene presence using southern
blot, how can you distinguish between the wild type and
transgenes?
Answer:
Specific restriction enzyme sites can be used - the use of different sites between
the transgene and endogenous gene will produce different band sizes
(depending where the sites are flanking) for comparison. The intensity of the
bands in the transgenes can also be used to determine the amount of copies
present when compared to the wild type.
Question:
9. What controls can be used during the southern blot to
determine transgene presence/quantity?
Answer:
Wild type (or no clone) DNA. This DNA will not produce a band for the transgene
restriction enzymes because the sites will not be present (negative control). The
band intensity for the wild type can also be used as a base line to compare the
intensity of the transgenes to (greater intensity than the wild type indicates more
gene copies)
, Question:
10. What is a reporter gene and how can be used in a
transgenic system?
Answer:
A reporter gene is an expression vector that is used in concert with a target
regulatory sequence that is used to study gene expression as opposed to gene
function. This is significant because transgenic mice in these systems can be used
to study changes in gene expression relative to changes in their promoters or
repressors.
Question:
11. How does creating a knockout mouse differ from
creating a transgenic mouse?
Answer:
A knockout mouse cannot have any copies of the target sequence while a
transgenic mouse can have multiple copies of an altered sequence. Therefore,
embryonic stem cells must be used to create knockout genes, via homologous
recombination, that can be passed on in the germ cell line.
Question:
12. What is the Tk gene in relation to the knockout selection
process?
Answer:
Thymidine kinase gene which results in sensitivity to ganciclovir. Normally,
mammalian cells are resistant to ganciclovir. If non-homologous recombination
occurs within the ES cells, they will be susceptible to ganciclovir.
Question:
13. Why does non-homologous recombination result in
ganciclovir susceptibility?
Answer:
The Tk gene is present on the vector plasmid containing the knockout of the
target gene. However, it is located outside of the target gene so it will not be
present during homologous recombination. If non-homologous recombination
occurs, a larger chunk of the vector will recombine containing the Tk gene.
QUESTIONS AND CORRECT ANSWERS
Question:
1. What does transgenic mean?
Answer:
Refers to an organism that has been genetically altered to contain additional DNA
(from the same organism or others) in order to alter expression of specific
phenotypes to compare with wild type expression
Question:
2. When creating a transgenic mouse, how many copies are
inserted and where will they insert into the genome?
Answer:
Between 1-200 copies of the transgene can be inserted into the genome at
random.
Question:
3. Which of the following statements is true about a
transgenic mouse?
Answer:
The transgenic mouse may have multiple copies of the transgene
3 multiple choice options
Question:
4. Where is the transgene injected into the mouse?
Answer:
Into a fertilized oocyte (DNA directly injected within the male pronucleus) or
embryo (using a retrovirus). The surviving embryos are then implanted back into
the donor female
Question:
5. Once offspring are obtained from the donor mouse, how
are they determined to have the transgene(s)?
Answer:
Through the use of PCR (using transgene specific primers) followed by southern
blotting and comparing the transgenic mice genes to the wild type (endogenous)
genes. This process can confirm transgene presence as well as the copy number
of the gene.
,Question:
6. When determining transgene presence using PCR, how
can you distinguish between the wild type and
transgenes?
Answer:
Specific primers can be used - one primer will be shared between the transgene
and the endogenous gene and one will be unique to the transgene/cloning
vector (another option would be to add one unique to the endogenous gene, if
possible, for hetero/homozygous comparisons). Using these primers should
create banding that can be used to identify the presence or absence of the
transgene.
Question:
7. What is used as a control for the PCR reactions for
determining wild type or transgene presence?
Answer:
An unrelated universal mouse gene. This gene will be a good negative control for
transgene presence because it will not react with primers specific to the
transgene/vector and not produce a band to showcase lack of contamination.
Additionally, primers for this gene can be used as a control for to illustrate lack of
contamination when comparing to the wild type because it will have banding with
the universal gene but not with the transgene primers.
Question:
8. When determining transgene presence using southern
blot, how can you distinguish between the wild type and
transgenes?
Answer:
Specific restriction enzyme sites can be used - the use of different sites between
the transgene and endogenous gene will produce different band sizes
(depending where the sites are flanking) for comparison. The intensity of the
bands in the transgenes can also be used to determine the amount of copies
present when compared to the wild type.
Question:
9. What controls can be used during the southern blot to
determine transgene presence/quantity?
Answer:
Wild type (or no clone) DNA. This DNA will not produce a band for the transgene
restriction enzymes because the sites will not be present (negative control). The
band intensity for the wild type can also be used as a base line to compare the
intensity of the transgenes to (greater intensity than the wild type indicates more
gene copies)
, Question:
10. What is a reporter gene and how can be used in a
transgenic system?
Answer:
A reporter gene is an expression vector that is used in concert with a target
regulatory sequence that is used to study gene expression as opposed to gene
function. This is significant because transgenic mice in these systems can be used
to study changes in gene expression relative to changes in their promoters or
repressors.
Question:
11. How does creating a knockout mouse differ from
creating a transgenic mouse?
Answer:
A knockout mouse cannot have any copies of the target sequence while a
transgenic mouse can have multiple copies of an altered sequence. Therefore,
embryonic stem cells must be used to create knockout genes, via homologous
recombination, that can be passed on in the germ cell line.
Question:
12. What is the Tk gene in relation to the knockout selection
process?
Answer:
Thymidine kinase gene which results in sensitivity to ganciclovir. Normally,
mammalian cells are resistant to ganciclovir. If non-homologous recombination
occurs within the ES cells, they will be susceptible to ganciclovir.
Question:
13. Why does non-homologous recombination result in
ganciclovir susceptibility?
Answer:
The Tk gene is present on the vector plasmid containing the knockout of the
target gene. However, it is located outside of the target gene so it will not be
present during homologous recombination. If non-homologous recombination
occurs, a larger chunk of the vector will recombine containing the Tk gene.
