Download Visualizing gene expression and function at the cellular level

Andreia FERREIRA
Supervised by Kamilla MALINOWSKA, Didier PICARD
Science III, Department of Cell Biology
Visualizing gene expression and function
at the cellular level
Introduction
The work allowed me to investigate the profile of protein and DNA expression. The expression of
protein can be detected by Western blot or by immunofluorescence using appropriate antibodies.
And luciferase assay enables understanding the regulation of protein expression. On the other hand,
PCR represents an important method to study DNA. These basic techniques are applied to study the
biological processes at molecular levels.
In all experiments we studied estrogen receptor α (ERα) that is a nuclear receptor activated by sex
hormone estradiol (E2). We investigated also the expression of Hsp90, a chaperone protein of ERα. It
stabilizes ERα and protects it from degradation.
Western Blot
Western blotting is a method to detect proteins with specific antibodies. Proteins are separated
according to their size by gel electrophoresis. The blot is a membrane, almost always made of
nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and
application of an electrical current induces the proteins in the gel to move to the membrane where
they adhere. The membrane is then a replica of the gel protein pattern, and is subsequently stained
with an antibody.
Expression of ERα in mammalian (HEK 293T) cells

HEK 293T cells were seeded into 6-well plate and transfected with plasmids carrying ERα
gene. We had four samples, two with non-transfected cells, and two with the transfected
cells.

After 48h cells were harvested and lysed with RIPA buffer. This solubilizes the proteins so
they can migrate individually through a separating gel.
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Andreia FERREIRA
Supervised by Kamilla MALINOWSKA, Didier PICARD
Science III, Department of Cell Biology

We added protease inhibitor cocktail to RIPA buffer to protect proteins from proteases that
are enzymes that break the peptide bonds that link amino acids together in the polypeptide
chain forming the protein.

The cells were incubated in RIPA buffer and they were put in the sonnicator to help for the
deployment/release of the proteins.

The samples were loaded on polyacrylamide gel. We used 10% of gel concentration because
studied proteins had low molecular weight.

After gel separation proteins were transferred to nitrocellulose membrane.
Results
The expression of ERα was detected by ERα
antibodies in Hek293T cells transfected with
ERα carrying plasmid. The expression of ERα
was not observed in non-transfected Hek293T
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Conclusions
ERα protein was expressed in HEK 293T cells.
Immunofluorescence
Immunofluorescence technique uses the specificity of antibodies to their antigen to target
fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of
the distribution of the target molecule through the sample.
Translocation of ERα into nucleus after E2 treatment

HeLa cells were seeded into 12-well plate and transfected with GFP-ERα and Flag-ERα
carrying plasmids. After 24h cells were treated with 100nM E2 for 90 minutes.

We had four samples: one with GFP-ERα without E2 treatment
One with GFP-ERα with E2 treatment
One with Flag-ERα without E2 treatment
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Andreia FERREIRA
Supervised by Kamilla MALINOWSKA, Didier PICARD
Science III, Department of Cell Biology
One with Flag-ERα with E2 treatment

Flag-ERα transfected HeLa cells: the cells were incubated with anti-Flag antibodies and
expression was detected by fluorescent microscope.

GFP-ERα transfected HeLa cells: expression was detected by fluorescent microscope.
Results
The expression of Flag-ERα
and GFP-ERα was observed
in transfected HeLa cells. In
samples treated with E2
ERα
GFP-ERα non-induced
GFP-ERα induced (E2)
was
detected
in
nucleus.
Conclusion
Flag-ERα non-induced
Flag-ERα induced (E2)
ERα accumulates in nucleus
after E2 treatment.
Luciferase assay
The Luciferase Assay System is an extremely sensitive assay for quantitation of gene expression.
Luciferase gene is controlled by regulatory element of
our gene of interest. The luciferase reagent is added to
cells expressing luciferase enzyme. The processed
reagent emits the light that could be detected.
Regulation of ERα-target gene expression

HEK 293T cells were seeded and transfected
with three plasmids:
o
Plasmid with firefly luciferase gene
controlled by estrogen response
elements (ERE)
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Andreia FERREIRA
Supervised by Kamilla MALINOWSKA, Didier PICARD
Science III, Department of Cell Biology
o
Plasmid with renilla luciferase gene for normalization
o
ERα gene

The cells were treated with 100nM E2

After 24h, the luciferase activity was measured.
Conclusion
The expression of firefly luciferase was activated after E2 treatment.
Genotyping
Genotyping is the process of determining differences in the genetic make-up (genotype) of an
individual by examining the individual's DNA sequence. It reveals the alleles an individual has
inherited from their parents.
Genetic mouse model for ERα chaperone protein Hsp90

Ear samples from mice were collected and PCR that is used to amplify a particular DNA
sequence (Hsp90) was performed


PCR procedure:
o
Denaturation: It separates the two stalks of the DNA from each other
o
Annealing: Taq polymerase (enzyme) catalyses DNA synthesis
o
Elongation: transcribed RNA is getting elongated
The PCR samples were loaded on agarose gel.
Results
Mice
n°1645
and
n°1647
expressed Hsp90 from only one
allele.
Conclusion
We found mice model with Hsp90
expression from one allele.
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Andreia FERREIRA
Supervised by Kamilla MALINOWSKA, Didier PICARD
Science III, Department of Cell Biology
Discussion
Molecular biology methods allow getting genetically modified cells by overexpression of genes from
plasmid. Then the overexpressed proteins can be studied: Their expression (here ERα expression) can
be detected (western blot, immunofluorescence), their activity can also be tested and their effect on
target genes (like ERα and luciferase gene with ERE) can be studied. We can also observe that after
ligand treatment proteins can translocate like ERα after E2 to nucleus. People work not only with
cells but also with living organisms like mice (genotyping).
Thanks
After a wonderful week in Cell Biology laboratories, I would like to thank Professor Didier Picard and
Kamilla Malinowska for all assistance and help, but also for all the time spent with me stirring up my
interest. In addition, I would like to thank “La Science appelle les jeunes” for giving me the
opportunity of spending an interesting week in a laboratory. For me, it’s memorable and I look
forward to my future studies around this domain.
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