Download Analytical techniques in developmental genetics A problem set Dr

Analytical techniques in developmental genetics
A problem set
Dr. Heather Verkade
Monash University
May 2013
As part of this resource you will find :
Part 1: Handout for students
Part 2: Instructor resource. Answers and notes as to the expectations for each question
Part 3: Answers for students.
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PART 1 : Handout for students
PRACTICAL PROBLEM SET:
ANALYTICAL TECHNIQUES IN DEVELOPMENTAL GENETICS
QUESTIONS:
1. You have identified three zebrafish mutants with point mutations in the otx gene, a gene
involved in early patterning of the vertebrate brain. They have identical phenotypes.
You let homozygous mutant embryos and wildtype embryos develop until midsomitogenesis. You have fixed them in batches of 50 homozygous mutant embryos (one
batch for each allele) and a batch of 50 wildtype embryos, and extracted RNA from the
batches. You have fixed another batch of each homozygous mutant and wildtype
embryos and extracted protein. You perform an RT-PCR on the mRNA samples, and a
western blot on the protein samples.
a. PCR is a procedure that amplifies DNA. What steps have been added to the
procedure to change it to RT-PCR, which will test for the presence of mRNA
instead?
b. What is a likely location of the mutation in the otx gene in allele otx1?
c. What is a likely location of the mutation in the otx gene in allele otx2?
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d. What is a likely location of the mutation in the otx gene in allele otx3?
e. The RT-PCR procedure starts with isolation of mRNA. This is then treated with
DNase to destroy the DNA. How would the RT-PCR result differ if the DNA had
not been removed successfully from the RNA sample from otx1/otx1 embryos?
f.
Sometimes RT-PCR produces two different sized bands for the one gene. What
might this indicate (in a wildtype)?
2. You have created a zebrafish transgenic with a GFP reporter gene designed to show
which cells express the krox20 gene.
a. Explain the design of the transgenic (the most basic type that gives this result)
with a diagram explaining the location of the various sequences in the construct.
b. You put the transgenic embryos on the fluorescent microscope and observe
expression of GFP in specific cells in hindbrain. Based on the sequence of the
krox20 gene, you suspect it might encode a transcription factor. Where would
you expect the Krox20 protein to be located within the cells?
c. How can you design a transgenic to determine the location of the Krox20 protein?
d. You take your krox20 transgenic fish, and you cross them together to generate
fish that are homozygous for the transgenic insertion. You inspect 115 embryos
and notice that 26 of them show a heart defect. These are also the embryos with
the strongest expression of the GFP. krox20 is not expressed in the heart. What
is causing this phenotype?
3. You have noticed that certain cells in the developing zebrafish neural tube (future brain
and spinal cord of a vertebrate) always divide in the same direction (ie, the direction of
the mitotic spindle is always in the same direction within the tissue). Some genes that
control asymmetric division in Drosophila cells are known, and you have determined that
these same genes exist in zebrafish.
a. What pattern of protein localization would you expect for a gene controlling
asymmetric division in one of these asymmetrically dividing cells?
b. What non-transgenic technique could you use to observe the localization of the
proteins in the cells of the neural tube?
c. What transgenic technique could you use to see the localization of the proteins in
the cells of the neural tube?
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4. Naturally occurring mutants were initially used in genetic studies (eg. Morgan’s white
mutation of Drosophila), but now mutagens are used to increase the frequency of
mutations you will find in your screen.
a.
Many loss-of-function mutations are recessive. Why?
b.
There are three ways in which a mutation could cause a dominant phenotype. What
are these three ways?
c.
What could cause a loss-of-function mutation to be dominant if the protein product
acts as a tetramer?
5. Consider a mouse gene, Zic2. You interbreed male and female mice heterozygote for a
loss of function allele of Zic2, and 30 pups are born. 21 of the offspring show a kinked
tail phenotype, and 9 show wild type phenotype.
a.
Explain this ratio. It may help to draw out a punnett square.
b.
You identify another mouse mutant that causes the same phenotypes. What is the
test you could do to determine if this mutation is in the Zic2 gene or another gene?
Describe with two crossing schemes, one for a mutation in the same gene, and one
for a mutation in another gene.
6. You determine the expression pattern of the twist-related gene in mice. You see that it is
expressed in the early developing structures of the limb, heart, somites (muscle blocks),
jaw and several other places. You have generated a knockout mouse, and the
homozygous knockout embryos show defects in somite formation and heart formation.
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a.
The limbs develop normally in twist-related homozygous mutants. What would you
conclude from this?
b.
You can’t examine jaw development in homozygous knockout twist-related mice
because they die from the heart defect before the jaw starts to form. What other type
of transgenic technique could you use to examine the role of twist-related in the
development of the mouse jaw? Explain how you would use this technique to
determine the role of twist-related in the jaw.
PART 2 : Instructor resource
ANALYSIS QUESTIONS PART 1:
More advanced answers or additional comments are given in parenthesis.
Question 1.
This question probes the students ability to interpret the presence/absence of mRNA and
protein, and thus predict where in the gene a mutation may lie. It addresses common
misconceptions such as that mutant alleles are never expressed, or that all mutations are
coding mutations.
a. The enzyme reverse transcriptase has been added to the RNA sample, so it can
generate the cDNA. A standard PCR is then performed using primers specific for otx.
b. As there is no transcription, I would predict a mutation in the promoter or an enhancer.
(A nonsense mutation may also cause this result due to nonsense-mediated decay.)
c. There is transcription but no translation, so this could have a mutation in the ribosome
binding site or in the ATG
d. Transcription and translation are normal, but this mutation has the same phenotype as
those with no protein product indicating that it is a null mutation. It is caused by a point
mutation in the coding region. (Other possible results are a normal sized mRNA product
with a smaller protein product due to a frameshift or a nonsense mutation, and a
different sized mRNA product and protein due to a mutation affecting splicing)
e. The PCR primers may prime from the genomic DNA so there would be a band in the
otx1/otx1 embryos, even though there is no transcription. (note that a technical method to
avoid this would be to design the primers so that they span an intron and therefore
produce a different size from the genomic, or so that they span an intron-exon boundary
and cannot bind to the genomic DNA)
f.
That there are multiple splice forms of this gene
Question 2.
This probes the ability of the students to differentiate between a reporter gene and a gene
fusion. Part d tests the misconception that the transgenic (which in a zebrafish is randomly
inserted) is changing the original gene, as the question requires the student to realize that
the transgene has inserted inside a gene with a totally different function.
Possible extensions to this are to introduce an example in which the reporter gene does not
recapitulate the full expression of the endogenous gene as seen by in situ hybridization or
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antibody stain. A question asking why this is the case would probe the students’
understanding of the nature of a promoter, and that the important enhancers may not be
within the selected ‘promoter’ sequence as they may be upstream, downstream or in an
intron.
a. The simplest approach would be a reporter gene with the krox20 promoter followed by
the coding sequence of the GFP gene.
krox20 promoter
GFP coding sequence
b. Since Krox20 is a transcription factor I would expect it to be within the nucleus of the
cells.
c.
krox20 promoter
krox20 coding sequence GFP coding sequence
You would design a gene fusion instead, so it has the krox20 promoter, then the krox20
coding sequence in frame with the GFP coding sequence. The protein that is produced
is a fusion of the Krox20 protein and GFP and should hopefully be transported to the
usual location of the Krox20 protein.
d. The transgene has inserted into a gene that functions in heart development. This is
effectively an insertional mutant. When you make it homozygous for the transgene, you
are making it homozygous for the insertion in the heart gene, and this is causing a
recessive phenotype.
Question 3.
This question is tailored to students studying developmental genetics and cell biology. It
relates to the localization of proteins, and asks the students to predict the localization of
proteins involved in asymmetric division based on information from other parts of the
course.
a. I would expect the protein localization to be asymmetric. For example, the protein may
be localized down one end of the cell at one end of the mitotic spindle such that when
cytokinesis occurs all the protein ends up in one daughter cell and not the other.
b. You could perform an antibody stain on wholemount embryos using an antibody that
recognizes the protein.
c. A gene fusion transgenic would allow you to see the localization of the protein.
Question 4.
This question revises the fundamentals of why an allele would be recessive or dominant.
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a. Because the wildtype allele can usually compensate for the mutant allele. In these
cases, one wildtype copy of the gene is sufficient.
b. Dominant negative, in which the mutant protein interrupts the function of the wildtype
protein. Happloinsufficiency, in which one copy of the gene does not produce
sufficient product for a wildtype phenotype. Gain of Function, in which the the
mutation has given the protein a new or different function (or caused the gene to be
expressed in different times/areas)
c. This can occur if all four subunits must be wildtype for the tetramer to function, and
the mutant protein can still bind with wildtype proteins to form a tetramer. This would
cause all of the tetramers with one, two, or three mutant proteins to be nonfunctional,
with only the tetramers containing 4 wildtype subunits able to function. As this is 1/16
of the tetramers, this dramatically reduces the functional tetramers, and could cause a
loss of function phenotype.
Question 5.
This question puts the concept of a pleiotropic mutation in context with a mouse mutation
that causes homozygous lethality and a heterozygous phenotype. The complementation
cross should be known to students, but has the added complexity of a pleiotropic mutation
with a recessive lethal phenotype.
It is advantageous for students to be familiar with the idea that the phenotype of a mouse
mutant embryo can be established by pre-natal analysis.
a. It looks like a 1:2:1 ratio, wildtype : kinked tails : lethal. The mice that are homozygous
for the Zic2 mutation have died and are not seen in the pups. Note that if a mouse
mutant dies in utero it is usually resorbed by the mother and so you don’t see the dead
ones in the litter.
b. This is a complementation cross, which is a little more complex to consider for a
recessive mutation. It is the recessive lethal phenotype which gives you the answer, and
so a first step would be to look at the embryos pre-natally to determine the phenotype
causing lethality. Then the complementation cross would be examined to determine if
this phenotype was present within the clutch.
Possible result 1: if the mutations are in the same gene:
Zic2+ / Zic2m1 x Zic2+ / Zic2m2
F1:
Zic2+ / Zic2+ = wildtype
Zic2+ / Zic2m1 = kinky tails
Zic2+ / Zic2m2 = kinky tails
Zic2m1 / Zic2m2 = lethal (would not be seen in pups)
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Possible result 2: if mutations are in different genes (I will call the second gene Zic3)
Zic2+/Zic2m Zic3+/Zic3+ x Zic2+/Zic2+ Zic3+/Zic3m
F1:
Zic2+/Zic2+ Zic3+/Zic3+ = wildtype
Zic2+/Zic2m Zic3+/Zic3+ = kinky tails
Zic2+/Zic2+ Zic3+/Zic3m = kinky tails
Zic2+/Zic2m Zic3+/Zic3m = kinky tails
Question 6:
This question exposes the common misconceptions that if a gene is expressed in a tissue it
must have a function there and that a loss of function mutation must cause a phenotype.
a. The gene may be expressed there but have no function there. Alternatively, it might
have a function that is redundant with the function of another gene.
b.
You would use the Cre-Lox system to generate a jaw specific knockout.
1st. Make a knockin allele of twist-related with LoxP sites flanking an important region of the
gene
2nd. Cross this mouse to a transgenic mouse expressing Cre under the control of a jaw
promoter.
promoter from a gene
expressed in the jaw
Cre coding region
CRE
LoxP
LoxP
endogenous twistrelated gene
knockin of a version of
the twisty gene with
loxP sites
In the jaw:
In all other tissues:
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Twistrelated
PART 3 : Answers for students
ANALYSIS QUESTIONS PART 1:
Question 1.
a. The enzyme reverse transcriptase has been added to the RNA sample, so it can
generate the cDNA. A standard PCR is then performed using primers specific for otx.
b. As there is no transcription, I would predict a mutation in the promoter or an enhancer.
(A nonsense mutation may also cause this result due to nonsense-mediated decay.)
c. There is transcription but no translation, so this could have a mutation in the ribosome
binding site or in the ATG
d. Transcription and translation are normal, but this mutation has the same phenotype as
those with no protein product indicating that it is a null mutation. It is caused by a point
mutation in the coding region. (Other possible results are a normal sized mRNA product
with a smaller protein product due to a frameshift or a nonsense mutation, and a different
sized mRNA product and protein due to a mutation affecting splicing)
e. The PCR primers may prime from the genomic DNA so there would be a band in the
otx1/otx1 embryos, even though there is no transcription. (note that a technical method to
avoid this would be to design the primers so that they span an intron and therefore
produce a different size from the genomic, or so that they span an intron-exon boundary
and cannot bind to the genomic DNA)
f.
That there are multiple splice forms of this gene
Question 2.
a. The simplest approach would be a reporter gene with the krox20 promoter followed by
the coding sequence of the GFP gene.
krox20 promoter
GFP coding sequence
b. Since Krox20 is a transcription factor I would expect it to be within the nucleus of the
cells.
c.
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krox20 promoter
krox20 coding sequence GFP coding sequence
You would design a gene fusion instead, so it has the krox20 promoter, then the krox20
coding sequence in frame with the GFP coding sequence. The protein that is produced
is a fusion of the Krox20 protein and GFP and should hopefully be transported to the
usual location of the Krox20 protein.
d. The transgene has inserted into a gene that functions in heart development. This is
effectively an insertional mutant. When you make it homozygous for the transgene, you
are making it homozygous for the insertion in the heart gene, and this is causing a
recessive phenotype.
Question 3.
a. I would expect the protein localization to be asymmetric. For example, the protein may
be localized down one end of the cell at one end of the mitotic spindle such that when
cytokinesis occurs all the protein ends up in one daughter cell and not the other.
b. You could perform an antibody stain on wholemount embryos using an antibody that
recognizes the protein.
c. A gene fusion transgenic would allow you to see the localization of the protein.
Question 4.
a. Because the wildtype allele can usually compensate for the mutant allele. In these
cases, one wildtype copy of the gene is sufficient.
b. Dominant negative, in which the mutant protein interrupts the function of the wildtype
protein. Happloinsufficiency, in which one copy of the gene does not produce sufficient
product for a wildtype phenotype. Gain of Function, in which the the mutation has given
the protein a new or different function (or caused the gene to be expressed in different
times/areas)
c. This can occur if all four subunits must be wildtype for the tetramer to function, and the
mutant protein can still bind with wildtype proteins to form a tetramer. This would cause
all of the tetramers with one, two, or three mutant proteins to be nonfunctional, with only
the tetramers containing 4 wildtype subunits able to function. As this is 1/16 of the
tetramers, this dramatically reduces the functional tetramers, and could cause a loss of
function phenotype.
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Question 5.
a. It looks like a 1:2:1 ratio, wildtype : kinked tails : lethal. The mice that are homozygous
for the Zic2 mutation have died and are not seen in the pups. Note that if a mouse
mutant dies in utero it is usually resorbed by the mother and so you don’t see the dead
ones in the litter.
b. This is a complementation cross, which is a little more complex to consider for a
recessive mutation. It is the recessive lethal phenotype which gives you the answer, and
so a first step would be to look at the embryos pre-natally to determine the phenotype
causing lethality. Then the complementation cross would be examined to determine if
this phenotype was present within the clutch.
Possible result 1: if the mutations are in the same gene:
Zic2+ / Zic2m1 x Zic2+ / Zic2m2
F1:
Zic2+ / Zic2+ = wildtype
Zic2+ / Zic2m1 = kinky tails
Zic2+ / Zic2m2 = kinky tails
Zic2m1 / Zic2m2 = lethal (would not be seen in pups)
Possible result 2: if mutations are in different genes (I will call the second gene Zic3)
Zic2+/Zic2m Zic3+/Zic3+ x Zic2+/Zic2+ Zic3+/Zic3m
F1:
Zic2+/Zic2+ Zic3+/Zic3+ = wildtype
Zic2+/Zic2m Zic3+/Zic3+ = kinky tails
Zic2+/Zic2+ Zic3+/Zic3m = kinky tails
Zic2+/Zic2m Zic3+/Zic3m = kinky tails
Question 6:
a. The gene may be expressed there but have no function there. Alternatively, it might
have a function that is redundant with the function of another gene.
b.
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You would use the Cre-Lox system to generate a jaw specific knockout.
1st. Make a knockin allele of twist-related with LoxP sites flanking an important region of the
gene
2nd. Cross this mouse to a transgenic mouse expressing Cre under the control of a jaw
promoter.
promoter from a gene
expressed in the jaw
Cre coding region
CRE
LoxP
LoxP
endogenous twistrelated gene
knockin of a version of
the twisty gene with
loxP sites
In the jaw:
In all other tissues:
12
Twistrelated