CRITICAL THINKING QUESTIONS 1 (Total: 25 marks)
Due May 26 by 5 pm EST
1. From the literature, you found out that Gene A is associated with liver cancer in humans but there is no
direct evidence of this. You want to test this in the lab using a model organism, but you find a homolog
(a gene with a similar sequence suggesting evolutionary conservation) of the human gene A in your
model. You are able to silence the expression of the homolog in the model and introduce the human
gene A as a transgene into the model organism (silencing the homolog will rule out the possibility that
the homolog is causing the cancer/malignant phenotype). In your construct that was introduced into
the model organism, you used a promoter that is conditionally expressed so that you can turn gene A
on and off when you need as you do not want the cancer phenotype to be expressed all the time.
a) What model can you use to study this? Explain your answer. (2 marks)
I would suggest using mice as a model organism. This is because they have many traits befitting a model
organism, and are relatively similar to human genes. They also have shorter life spans and their genomes have
been well-studied.
b) Using a model organism, you are trying to find out if expression of gene A is causing the malignant
phenotype associated with liver cancer in humans. How can you test this? What approach will you
take to investigate this? (4 marks)
1. You can compare your mutant models to the wild-type organisms. By assessing the phenotypic
differences between the two, and observing to see if the model organism develops any cancerous
traits such as cell malignancy, you will be able to conclude that gene A is the causative agent. If the
wild-type remains unchanged while the mutant would develop the cancer phenotype.
2. To compare the two, you could, as aforementioned, look for any deformations or signs of malignancy.
You could also observe any tumor formations and compare different lung tissues to identify possible
effects.
c) Do you need any controls for this experiment? If yes, what can be a positive and a negative
control? (4 marks)
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-Yes, of course you would require controls. There may be conditional genetic or environmental
factors affecting your results.
Positive: You could use other model organisms with known and proven liver cancer genes in the
same conditions, to prove the conditions of the experiment are not limiting.
Negative: Include a group of model organisms possessing the silent homolog but not the
introduced transgene. This will validate the functionality of the silenced homolog on the
malignancy
d) If you find that A is not causing the malignant phenotype, what will be the next logical step to look
for other candidate genes that might be causing the phenotype? Will you use humans or the model
to do this experiment? What approach will you take, and which molecular species will you choose
to look at? Explain in detail. We have not discussed any techniques yet to perform experiments
related to this but have talked about approaches to address the problem. Base your answers on the
approaches and not the techniques. Do not search the web and come up with ideas and techniques
that we have not covered yet. Please stick to the contents which we have covered as otherwise,
this exercise will not be helpful. (4 marks)
If gene A is found to not be the cause of the cancerous phenotype, the logical next step would be to consult
other literature to discover separate genes that may be typically associated with lung cancer. If none can be
found, Genomic Analysis would be a good way to begin researching possible related genes on your own.
I would use humans, given we are applying Forward Genetics principles and do not know exactly which gene
we are looking for- the reason model organisms worked in the prev. experiment was because we had already
identified a possible gene and found a similar gene in the model organism.
To find genes of interest, I would first Screen for mutants that exhibit the phenotype of interest. In this case, it
is lung cancer. I would then perform Untargeted genetic profiling in mutant tumors in order to analyze
possible linked genes.
The molecular species I would use could be Transcripts, given I could look for altered sequences, abundance of
transcripts and edits to the transcription in a gene. These could all be factors causing the variance. Genetic
Material could also be studied.
e) Can you suggest one control for this experiment? (2 marks)
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I would compare the wild-type, control species to the mutant experimental species to ensure that our results
are valid. In doing so we can verify our genes of interest are unique to the mutants.
f) What can be a logical next step after you have found a number of molecular species that were
present or absent or over or under-expressed in the tumor? (3 marks)
After identifying molecular species of interest in the tumor, you could utilize the scientific method to
formulate and develop a hypothesis. You can then design an experiment, and its controls, where you
would be able to prove your hypothesis. This could be by removing the gene of interest from a mutant and
seeing if it still develops the cancerous phenotype or not.
2. Mention one experiment where you will need to use transcriptional fusion and another experiment
where you will need to use translational fusion. Again, do not search the web to get your answer.
Understand the techniques and come up with your own experiments. (6 marks)
Transcriptional: In a hypothetical situation where, for example, I was able to identify a mammalian species
whose front legs were considerably weaker than their hind legs, I could use transcriptional fusion to test a
hypothesis that perhaps gene expression is varying depending on the body’s region. By encoding the
promoter region with a reporter gene, we could identify whether gene expression were active in those
weaker areas!
Translational: You could use Translational Fusion to identify proteins and their localization methods. For
example, if there is a protein sequence that is abundantly expressed in a particular organelle, by attaching
that sequence to a reporter gene, you would be able to see how exactly the protein sequence targets and
ends up in that organelle.
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