Name_______________________________
Before we begin remind yourself what transcription factors and fibroblasts are:
What are transcription factors?
Transcription factors are proteins that bind to DNA and determine whether the DNA sequence is
transcribed into mRNA (and then later translated or formed into proteins). When a DNA
sequence is transcribed into mRNA, we say that the gene is “turned on,” or expressed.
Transcription factors are important because they regulate what proteins the cell is manufacturing
or making. Proteins are the worker bees of a cell, so what kind of proteins (kind of worker bee)
are being created determine how the cell is functioning and responding to external (or internal)
signals (stimuli).
What is a fibroblast?
A fibroblast is a type of cell that synthesizes the extracellular matrix (ECM), which is composed
of connective tissues such as collagen. The ECM provides the structural framework for animal
tissues, and plays a critical role in wound healing. Fibroblasts are the most common cell type
found in mammalian connective tissue.
Transcription Factors in Cell Reprogramming
Scientists have discovered that somatic cells or body cells can be directly reprogrammed to an
alternative differentiated fate without first becoming stem cells. For instance, body cells such as
fibroblasts can be reprogrammed into cardiomyocytes (heart muscle cells). Specific transcription
factors can be introduced to cells for reprogramming. The question lies in which transcription
factors allow this process to occur?
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This phenomenon of cell
reprogramming can be researched with
the aid of fluorescence. A cardiacspecific gene can be engineered to
fluoresce once it is “turned on”
(expressed) by the transcription factors
that allow reprogramming to occur. If
the fibroblasts that have been exposed
to the necessary transcription factors,
they will properly differentiate into
heart muscle cells, and the cardiac
muscle gene will be get turned on
which can be seen by fluorescence
microscopy.
We know that fluorescence is a way of marking specific genes or factors in a cell to make them
glow which can be seen under a microscope. Think of some other examples that fluorescence can
be useful. What kinds of processes or cell structures would be helpful to see with fluorescence?
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Scientists initially identified all of the transcription factors that are associated with heart muscle
cell development. In total, five transcription factors were found (called transcription factors A, B,
C, D, and E). All five transcription factors were introduced into cultured body cells (cells grown
in a dish) to observe cell differentiation. They specifically used fibroblasts as the type of body
cells. The scientists wished to determine if all five transcription factors were necessary for the
reprogramming of fibroblasts into heart muscle cells, or if only some of them were needed. To
do so, they tried various combinations of transcription factors to identify if the variation of
specific transcription factors decreased fluorescent cardiac gene expression compared to when all
the transcription factors were used. If the removal a transcription factor decreased fluorescence,
the scientists could conclude it was required for reprogramming.
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The first experiment involved injecting fibroblasts with all of the transcription factors related to
cardiac muscle development. From this scientists, found that 13 million cells out of 20 million of
the cells fluoresced when all of the transcription factors were used. Then cells containing various
combinations of transcription factors were injected to see how many cells fluoresced.
A.Draw a picture showing the process of injecting fibroblasts with the transcription
factors and what type of cell is formed when the heart muscle gene is expressed.
Label the heart muscle cells with fluorescence. Cut out the pictures that are
provided to make a flow chart of what is happening.
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B. A total of 20 million cells were used for each trial. The following data displays the
amount of fluorescence seen for 3 trials in the new cells when various combinations of
transcription factors were added to the fibroblasts. Each of the trials for each of the
transcription factor combinations were done in separate petri dishes of 20 million cells.
Determine the average number of fluorescing cells in the 3 trials.
Transcription Factor
(TFs) Combinations
Number of
fluorescing cells
Trial 1 (millions of
cells)
Number of
fluorescing
cells Trial 2
(millions of
cells)
Number of
fluorescing
cells Trial 3
(millions of
cells)
B, C, D, E
12.3
12.1
12.4
A, C, D, E
1.6
1.8
2.0
A, B, D, E
15.3
14.6
15.9
A, B, C, E
1.1
1.2
1.3
A, B, C, D
0.96
1.0
0.98
All 5 TFs present (A,
B, C, D, E)
Control (no TFs)
13.1
13.0
12.9
0
0
0
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Average
number of
fluorescing
cells
(millions of
cells)
1. What type of graph would best represent the average data? Draw below
If the amount of cardiac gene fluorescence significantly decreases when a transcription factor is
removed compared to when all five transcription factors are used, the scientists conclude that
that transcription factor is necessary for reprogramming.
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2. After analyzing the graph and the data, which transcription factor combination(s) produce
the most cardiac muscle cells?
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3. According to your graphical analysis which specific transcription factor can be excluded?
Explain why.
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C. From the previous graphical analysis scientists determined that transcription factors C
and possibly A are unnecessary since the combinations without these transcription factors
produced the most heart muscle cells. When these transcription factors were present in
the combination the amount of heart muscle cells produced decreased. To be sure of their
results scientist will use a statistical test to prove that their results can be accepted.
Scientists will use a statistical test to determine that when transcription factor C and
possibly A are present in the combination, the number of heart muscle cells produced is
significantly less compared to when these transcription are removed.
1. What is the most appropriate statistical test to use? Why would you use this test?
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2. What are the null and alternative hypothesis?
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3. Use an unpaired t test to determine that when transcription factors A and C are present in
the combination the amount of heart muscle cells produced is significantly less compared
to when they are removed. Remember that a p value of 0.05 or less is scientifically
accepted.
Fill in the following tables.
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Transcription Factor
Combination
Trial 1
(𝑥 − 𝑥̅ )2
Trial 2
(𝑥 − 𝑥̅ )2
Trial 3
(𝑥 − 𝑥̅ )2
Total Sum =
∑(𝑥 − 𝑥̅ )2
B, C, D, E
A, C, D, E
A, B, D, E
A, B, C, E
A, B, C, D
All 5 TFs present (A, B, C, D,
E)
Transcription
Factor (TFs)
Combinations
T Value
(compared to
when all 5 TFs
are added)
Null
Hypothesis
(Ho)
Degrees of
Freedom
(n – 1)
P Value
(compared to
accepted
significance)
B, C, D, E
A, C, D, E
A, B, D, E
A, B, C, E
A, B, C, D
4. After the statistical test what can you conclude about the transcription factor
combinations? Which transcription factor combination(s) is not necessary for making the
cardiac gene?
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5. According to your analysis which specific transcription factor(s) can be excluded?
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6. Which of the following specific transcription factor(s) are then necessary for the cardiac
gene to be expressed?
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7. When transcription factors A or C were present in the combinations, some (7-15%)
fluorescent cells were produced indicating that the cells did develop into the heart muscle
cells however not more than when all of the transcription factors were added or when
they were removed. Explain why this is so?
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D. A final experiment was conducted with only the three remaining transcription factors
Numberof Fluorescing Cells (millions of
cells)
scientists believe are needed for the reprogramming of fibroblasts into heart muscle cells.
Is these transcription factors are necessary for the expression of the cardiac gene, then if
one of the three transcription factors is removed there should be significantly less
fluorescence compared to when all three transcription factors are used. A total of 5 trials
were conducted for each of the transcription factor combinations. Each trial contained a
total of 1 million cells. A statistical test can be used to deem the findings scientifically
accepted. Determine if the results of the experiment are significantly less compared to
when all three transcription factors are used.
Average Number of cells fluorescing during various
combinations of transcription factors
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
B, D
B, E
D, E
Trancription Factor Combinations
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All 3 TFs present (B, D, E)
Transcription Number of
Factor (TFs) fluorescing
Combinations cells Trial
1 (millions
of cells)
Number of
fluorescing
cells Trial
2 (millions
of cells)
Number of
fluorescing
cells Trial
3 (millions
of cells)
Number of
fluorescing
cells Trial
4 (millions
of cells)
Number of
fluorescing
cells Trial
5 (millions
of cells)
B, D
0.22
0.21
0.22
0.24
0.24
B, E
0.19
0.18
0.17
0.19
0.19
D, E
0.24
0.28
0.25
0.26
0.23
All 3 TFs
0.78
present (B, D,
E)
Control (no
0
TFs)
0.82
0.83
0.65
0.73
0
0
0
0
Average
number of
fluorescing
cells
(millions
of cells)
1. What is the most appropriate statistical test to use? Explain why.
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2. What are the null and alternative hypothesis?
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3. Use an unpaired t test to determine that when transcription factors A and C are present in
the combination the amount of heart muscle cells produced is significantly less compared
to when they are removed. Remember that a p value of 0.05 or less is scientifically
accepted.
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Fill in the following tables.
Transcription Factor Trial 1
Combination
(𝑥 − 𝑥̅ )2
B, D
Trial 2
(𝑥 − 𝑥̅ )2
Trial 3
(𝑥 − 𝑥̅ )2
Trial 3
(𝑥 − 𝑥̅ )2
Trial 5
(𝑥 − 𝑥̅ )2
Total Sum =
∑(𝑥 − 𝑥̅ )2
B, E
D, E
All 3 TFs present
(B, D, E)
Transcription Factor
(TFs) Combinations
T Value
Null
(compared to
Hypothesis
when all 3 TFs are (Ho)
added)
Degrees of
Freedom
(n – 1)
P Value
(compared to
when all 3 TFs are
added)
B, D
B, E
D, E
4. What can you conclude from the data analysis?
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