MAMMALIAN GENETICS BCH5413 EXAM 2
WITH COMPLETE QUESTIONS AND
ANSWERS
Foreign DNA is inserted into the genome - ANSWER What is the
meaning of the term transgenic?
The transgenic mouse is screened to confirm the transgenic gene is
present, then it is crossed with another transgenic mouse to start a
line of transgenic mice - ANSWER Once an oocyte has been injected
with a transgene and implanted into a mouse, what are the steps to
obtaining offspring?
True (this integration is random) - ANSWER T/F: the transgene is
randomly integrated into a mouse genome.
Gain - ANSWER The goal of creating a transgenic mouse is to study
___ of function.
Loss - ANSWER The goal of creating a knockout mouse is to study ___
of function.
True (the transgene will cause overexpression by increasing copy
number of the gene) - ANSWER T/F: the mouse that a transgene is
being added to already has 2 copies of that gene.
(1) PCR: SP6 and T7 primers will only amplify the transgene (bac
vector) and beta-globin primers will only amplify the endogenous
gene; (2) Southern blot: probes specific to either the endogenous
gene or transgene are used - ANSWER Once an oocyte has been
injected with a transgene and implanted into a mouse, what are the 2
possible ways to determine which offspring have the transgene?
False (SP6 and T7 sites are only found in the bac vector, not the
,endogenous gene) - ANSWER T/F: in a gel of PCR of a transgenic
mouse that HASN'T been injected with the bac vector, there should
be bands for both the SP6 and T7 primers.
True (beta-globin is only present in the genome, not in the bac
vector) - ANSWER T/F: in a gel of PCR that HASN'T been injected with
the bac vector, there should only be a band for beta-globin.
The gene of interest is cloned into a bac vector, microinjected into a
mouse embryo, and transplanted into a female mouse who will carry
the embryo to term - ANSWER How is the transgene incorporated
into a mouse genome?
By using probes specific to either the endogenous gene or the
transgene - ANSWER In transgenic mice, how could you determine
between the endogenous gene and transgene in a Southern blot?
To determine in which tissue a regulatory gene might control gene
expression - ANSWER What would be the purpose of creating a
reporter gene as a transgene?
"Where in the mouse is this protein normally expressed?" - ANSWER
What type of experimental question could you answer by creating a
reporter gene as a transgene?
lacZ gene - ANSWER What gene is the reporter gene inserted before
in a plasmid for a reporter gene construct?
So it can easily realign with the mouse chromosome upon injection -
ANSWER Why does the plasmid for a reporter gene construct need to
be LINEAR before microinjection?
Disrupted (typically by the neomycin resistance gene) - ANSWER In
knockout mice, the gene is:
In a knockout mouse line, the genome itself is altered (unlike a
transgenic mouse line where extra genes are added), so stem cells
must be used - ANSWER Why is the process for creating a knockout
mouse different than creating a transgenic mouse?
,The ES cells need to be injected with the gene of interest disrupted
by the neomycin resistance gene to allow for selection of the correct
cell line after microinjection - ANSWER Why do embryonic stem (ES)
cells need to be engineered in creating a knockout mouse?
In the middle of the gene of interest (making it nonfunctional) -
ANSWER Where is the neomycin resistance gene located in the
disrupted gene of interest?
Tk (thymidine kinase) gene - ANSWER What gene causes cells to be
sensitive to ganciclovir?
Cells sensitive to neomycin didn't get injected with the gene of
interest (they don't have neomycin resistance and will die off); cells
sensitive to ganciclovir contain the Tk gene (the plasmid was injected
in the wrong location, bringing the Tk gene with it) - ANSWER When
creating knockout mice, why do cells need to be isolated that are
sensitive to G418 (neomycin) AND ganciclovir?
True (random recombination means it wasn't inserted into the
correct location) - ANSWER T/F: homologous recombination means
that the plasmid was inserted into the correct location in the mouse
genome.
False (the Tk gene is only inserted in random recombination) -
ANSWER T/F: homologous recombination means the Tk gene was
inserted along with the gene of interest.
False (only brown mice will have the knockout gene because it was
injected into their ES cells) - ANSWER T/F: black mice can contain the
knockout gene.
(1) Black mouse with WT gene; (2) brown mouse with WT gene; (3)
brown mouse that is heterozygous for the knockout gene; offspring
can be created by crossing 2 of the brown heterozygous mice -
ANSWER What are the 3 possible offspring that can be produced
from a chimeric (knockout) mouse and a black (WT) mouse? How will
offspring be created?
, 25% - ANSWER If 2 brown mice heterozygous for the knockout gene
are crossed, what % of their offspring will be homozygous for the
knockout gene?
False (black coat color is recessive and brown coat color is
dominant) - ANSWER T/F: the black coat color in mice is dominant.
The WT mouse will have a band that corresponds to the EcoRI cut
sites on either end of the gene; the heterozygous mouse will have 2
bands that correspond to the 3 EcoRI cut sites (2 on either side of the
gene and 1 in the middle of the knockout gene) - ANSWER In a
Southern blot of a knockout mouse's DNA, why does the WT mouse
have 1 band and the heterozygous mouse have 2 bands?
(1) The gene can be knocked out AFTER initial development,
preventing fatal knockouts from preventing development; (2) the
gene can be knocked out only in CERTAIN tissues - ANSWER What
are the 2 advantages of creating a Cre/Lox knockout mouse over the
traditional knockout mouse?
2 different loxP sites flank exon 2 of the target gene (to be knocked
out) - ANSWER Where in the genome are loxP sites located in a
Cre/Lox model?
The loxP mouse functions normally until it's crossed with a cre mouse
-> its offspring will express both the target gene flanked by loxP sites
and the cre protein, which will knockout the gene when the cre
promoter becomes activated - ANSWER Why does the loxP mouse
need to be crossed with the Cre mouse to create a functional
system?
The promoter before the Cre gene (the promoter can be cell-specific,
causing Cre to only be produced in certain cells/tissues) - ANSWER
What in the Cre/Lox system allows the gene to be knocked out only
under specific conditions?
The process of removing the nucleus from a somatic (body) cell and
injecting it into an egg cell (nucleus removed) -> the egg cell
becomes pluripotent and can be placed into a surrogate mother or
WITH COMPLETE QUESTIONS AND
ANSWERS
Foreign DNA is inserted into the genome - ANSWER What is the
meaning of the term transgenic?
The transgenic mouse is screened to confirm the transgenic gene is
present, then it is crossed with another transgenic mouse to start a
line of transgenic mice - ANSWER Once an oocyte has been injected
with a transgene and implanted into a mouse, what are the steps to
obtaining offspring?
True (this integration is random) - ANSWER T/F: the transgene is
randomly integrated into a mouse genome.
Gain - ANSWER The goal of creating a transgenic mouse is to study
___ of function.
Loss - ANSWER The goal of creating a knockout mouse is to study ___
of function.
True (the transgene will cause overexpression by increasing copy
number of the gene) - ANSWER T/F: the mouse that a transgene is
being added to already has 2 copies of that gene.
(1) PCR: SP6 and T7 primers will only amplify the transgene (bac
vector) and beta-globin primers will only amplify the endogenous
gene; (2) Southern blot: probes specific to either the endogenous
gene or transgene are used - ANSWER Once an oocyte has been
injected with a transgene and implanted into a mouse, what are the 2
possible ways to determine which offspring have the transgene?
False (SP6 and T7 sites are only found in the bac vector, not the
,endogenous gene) - ANSWER T/F: in a gel of PCR of a transgenic
mouse that HASN'T been injected with the bac vector, there should
be bands for both the SP6 and T7 primers.
True (beta-globin is only present in the genome, not in the bac
vector) - ANSWER T/F: in a gel of PCR that HASN'T been injected with
the bac vector, there should only be a band for beta-globin.
The gene of interest is cloned into a bac vector, microinjected into a
mouse embryo, and transplanted into a female mouse who will carry
the embryo to term - ANSWER How is the transgene incorporated
into a mouse genome?
By using probes specific to either the endogenous gene or the
transgene - ANSWER In transgenic mice, how could you determine
between the endogenous gene and transgene in a Southern blot?
To determine in which tissue a regulatory gene might control gene
expression - ANSWER What would be the purpose of creating a
reporter gene as a transgene?
"Where in the mouse is this protein normally expressed?" - ANSWER
What type of experimental question could you answer by creating a
reporter gene as a transgene?
lacZ gene - ANSWER What gene is the reporter gene inserted before
in a plasmid for a reporter gene construct?
So it can easily realign with the mouse chromosome upon injection -
ANSWER Why does the plasmid for a reporter gene construct need to
be LINEAR before microinjection?
Disrupted (typically by the neomycin resistance gene) - ANSWER In
knockout mice, the gene is:
In a knockout mouse line, the genome itself is altered (unlike a
transgenic mouse line where extra genes are added), so stem cells
must be used - ANSWER Why is the process for creating a knockout
mouse different than creating a transgenic mouse?
,The ES cells need to be injected with the gene of interest disrupted
by the neomycin resistance gene to allow for selection of the correct
cell line after microinjection - ANSWER Why do embryonic stem (ES)
cells need to be engineered in creating a knockout mouse?
In the middle of the gene of interest (making it nonfunctional) -
ANSWER Where is the neomycin resistance gene located in the
disrupted gene of interest?
Tk (thymidine kinase) gene - ANSWER What gene causes cells to be
sensitive to ganciclovir?
Cells sensitive to neomycin didn't get injected with the gene of
interest (they don't have neomycin resistance and will die off); cells
sensitive to ganciclovir contain the Tk gene (the plasmid was injected
in the wrong location, bringing the Tk gene with it) - ANSWER When
creating knockout mice, why do cells need to be isolated that are
sensitive to G418 (neomycin) AND ganciclovir?
True (random recombination means it wasn't inserted into the
correct location) - ANSWER T/F: homologous recombination means
that the plasmid was inserted into the correct location in the mouse
genome.
False (the Tk gene is only inserted in random recombination) -
ANSWER T/F: homologous recombination means the Tk gene was
inserted along with the gene of interest.
False (only brown mice will have the knockout gene because it was
injected into their ES cells) - ANSWER T/F: black mice can contain the
knockout gene.
(1) Black mouse with WT gene; (2) brown mouse with WT gene; (3)
brown mouse that is heterozygous for the knockout gene; offspring
can be created by crossing 2 of the brown heterozygous mice -
ANSWER What are the 3 possible offspring that can be produced
from a chimeric (knockout) mouse and a black (WT) mouse? How will
offspring be created?
, 25% - ANSWER If 2 brown mice heterozygous for the knockout gene
are crossed, what % of their offspring will be homozygous for the
knockout gene?
False (black coat color is recessive and brown coat color is
dominant) - ANSWER T/F: the black coat color in mice is dominant.
The WT mouse will have a band that corresponds to the EcoRI cut
sites on either end of the gene; the heterozygous mouse will have 2
bands that correspond to the 3 EcoRI cut sites (2 on either side of the
gene and 1 in the middle of the knockout gene) - ANSWER In a
Southern blot of a knockout mouse's DNA, why does the WT mouse
have 1 band and the heterozygous mouse have 2 bands?
(1) The gene can be knocked out AFTER initial development,
preventing fatal knockouts from preventing development; (2) the
gene can be knocked out only in CERTAIN tissues - ANSWER What
are the 2 advantages of creating a Cre/Lox knockout mouse over the
traditional knockout mouse?
2 different loxP sites flank exon 2 of the target gene (to be knocked
out) - ANSWER Where in the genome are loxP sites located in a
Cre/Lox model?
The loxP mouse functions normally until it's crossed with a cre mouse
-> its offspring will express both the target gene flanked by loxP sites
and the cre protein, which will knockout the gene when the cre
promoter becomes activated - ANSWER Why does the loxP mouse
need to be crossed with the Cre mouse to create a functional
system?
The promoter before the Cre gene (the promoter can be cell-specific,
causing Cre to only be produced in certain cells/tissues) - ANSWER
What in the Cre/Lox system allows the gene to be knocked out only
under specific conditions?
The process of removing the nucleus from a somatic (body) cell and
injecting it into an egg cell (nucleus removed) -> the egg cell
becomes pluripotent and can be placed into a surrogate mother or
