Download Visualizing gene expression and function at the cellular level

Elisabetta Erriu, Liceo Lugano 1
Week Report- Swiss Youth Science
Tutor: Dr Marta Madon-Simon
Prof. Didier Picard’s Laboratory, Department of Cellular Biology, University of
Geneva.
Visualizing gene expression and function at the cellular
level
Abstract:
During this week of study, we have used several techniques to investigate the
protein and gene expression in cells and tissues.
Introduction:
To visualize how gene are expressed and what are their functions, first it is
important to understand what is a gene.
Genes are portions of the DNA that serve as the basic units of heredity and
provide basis for building proteins, which perform important tasks for the cell
functions or serve as building blocks. The information contained within genes
determines the protein composition and therefore the functions of the cell. Every cell of the organism must contain the same genetic information and the
DNA is therefore duplicated before a cell divides: this process is called
replication. When in the organism specific proteins are needed, the
corresponding genes are transcribed into RNA: this process is called
1 transcription. The RNA is first processed so that non-coding parts are
removed and it is then transported out of the nucleus. Outside of the nucleus,
the proteins are built based upon the code in the RNA: this process is called
translation.
Figure 1: Illustration of DNA.
This figure illustrates the DNA of a cell (on the right). Inside the cell there is
the nucleus (on the left), which contains chromosomes. Chromosomes are
assembled by genes, that are specific portions of DNA.
DNA consists of two long polymers of simple units called nucleotides, with
backbones made of sugars and phosphate groups joined by ester bonds.
Attached to each sugar is one of four types of molecules called nucleobases
(in the figure with the letters C,T,G,A).
https://healthfinder.gov/FindServices/Organizations/Organization.aspx?code=
HR3150
Materials and methods:
To study how and when genes are expressed in various tissues or cells of
different organisms, scientists use different techniques.
In our project, we used the four main techniques that allowed us to visualize
gene expression and function.
2 1. Luciferase Assay:
• The luciferase assay is a technique commonly used as a tool to
study gene expression at the transcriptional level as well as other
cellular components and events that are involved in gene
regulation.
• In our experiment we wanted to understand how the Estrogen
Receptor (ER) and the Glucocorticoid Receptor (GR) regulate
gene expression, therefore we transfected cells with plasmids
coding for their expression and later treated these cells with
Dexamethasone (DEX) and Estradiol (E2), the hormones that
bind these receptors.
• Results of the experiment:
C: vehicle; E2: Estradiol (steroid hormone); Dex: Dexamethasone
(synthetic steroid hormone)
ERalpha Fold 20 15 10 ERalpha 5 0 C E2 DEX Figure 2: This graph shows the results of the Luciferase Assay experiment
and it is possible to see the activation of ERalpha gene after treatment with
E2, Dex or just vehicle. For this gene, as expected, the activation is highest
with the E2 treatment.
3 GR 8 Fold 6 4 GR 2 0 C E2 DEX Figure 3: This graph shows the results of the Luciferase Assay experiment
and it is possible to see the activation of GR gene after treatment with E2,
Dex or vehicle. For this gene, the activation is highest with the Dex treatment.
The two graphs show, as expected, that ER is activated by E2 and not by
Dex; on the other hand GR is regulated by Dex and not by E2.
2. Immunohistochemistry:
• Immunohistochemistry (IHC) is a wide-used biological technique that
allows for visualizing the localization of specific proteins in tissue
sections.
• In this case, we wanted to visualize the distribution and the
localization of a specific protein, called p23, in skin of wild type and
p23 knockout (lacking p23 protein) mice.
We performed IHC on paraffin sections, and the protocol that we
used was as follows:
Step 1: Deparaffinization and rehydration of the sections in ethanol
gradient solutions.
Step 2: Antigen Retrieval:
Sections were heated in the microwave in 10mM pH6 sodium citrate
-Tween 0.05% for antigen retrieval: 900W for 1min followed by 270W
for 20 min. Sections were then cooled down for 45-60 minutes on the
shaker at room temperature and finally washed in PBS.
Step 3: Quenching endogenous tissue peroxidase:
-Wax pen was used to draw around the sections (all the following
steps are performed in a humid box) and endogenous peroxidase
was quenched with 50% Methanol and 3% H2O2 in PBS for 20
minutes.
Step 4: Blocking:
4 -Sections were blocked for 1 hour at room temperature with the
appropriate blocking buffer (serum derived from the species that the
secondary antibody was raised in, in our case goat).
Step 5: Incubation of the sections with primary antibody:
-Sections were incubated O/N with primary antibody at 4oC.
Step 6: Washing the sections with secondary antibody:
-Sections were washed with PBS 0.1% Tween 20 three times for
10min and then incubated in humid box with biotinylated secondary
antibody for 45 min. At the end they were washed in PBS 0.1%
Tween 20 three times for 10 min
Step 7: ABC solution:
-First, sections were incubated with ABC elite kit (Vector labs) for 45
minutes at room temperature, in the humid box and then washed in
PBS 0.1% Tween 20 three times for 10min.
Step 8 : Reveal :
-DAB solution was applied and color was developed for as long as
needed, in our case reaction was stopped in mqH2O after 10 min.
Step 9: Counterstaining:
- Sections were incubated in hematoxylin for 15-30 seconds and
then rinsed under tap water.
Step 10: Sections were dehydrated in ethanol gradient solutions
and mounted.
5 Figure 4: This figure shows some major steps of IHC, including sample
preparation, antigen retrieval, as well as antibody staining.
1. Deparaffinisation: prompt and adequate tissue preparation is crucial in
immunohistochemistry.
2. Antigen retrieval: pre-treatment with the antigen retrieval reagent is needed
to break the protein cross-links formed by formalin fixation in order to uncover
antigen sites.
3. Blocking unoccupied sites
4. Adding primary Antibodies (necessary for the detection of protein of
interest)
5. Adding secondary Antibodies (binds to the primary Antibody).
http://www.ptglab.com/Support/TechnicalSupport/LearningCenter/Immunohist
ochemistry.aspx
• Results of the experiment:
p23IHConE18.5skinsamples
p23JJ3Ab,1:1000
Figure 5: Results of IHC on skin samples from wild type and p23 knockout
mice.
These photos shows three bright field microscope pictures of mouse skin :
the first row represents the skin of a wild type mouse (phenotype of a typical
6 form of species) stained with antibody for p23. The second row represents
the skin of a knockout mouse stained with anti-Body for p23. The last row
represents the skin of a wild type mouse without antibody for p23 which
served as our negative control.
In fact, the presence of p23 protein is confirmed in wild type skin for brown
staining in different parts of the skin tissue (for example basal keratinocytes).
In the knockout skin, p23 protein is not present and it is the reason why the
skin has no brown staining.
3. Western Blot:
•
Western blot is a technique that allows investigators to determine the
presence of a protein and to measure relative amounts of the protein
present in different samples.
•
The first step in a Western blotting procedure is SDS-PAGE, which
allows for the separation of proteins. Then the separated proteins are
transferred or blotted onto a nitrocellulose membrane. Next, the
membrane is blocked to prevent any nonspecific binding of antibodies
to the surface of the membrane.
The blocked membrane is probed with a primary antibody that
recognizes a specific protein, but it is not directly detectable. Therefore,
enzyme-conjugated secondary antibodies are used as the means of
ultimately detecting the target antigen.
Figure 6: This figure shows the detection in Western Blot.
Detection is one of the main and final steps of a Western Blot. The aim of this
step is to detect the signal due to presence of the protein of interest.
http://www.leinco.com/general_wb
7 Figure 7: Results of our Wesertn Blot experiment showing expression of ER
and GR proteins in transfected HEK cells.
In our experiment we wanted to visualize the presence of three proteins: GR,
ER and GAPDH (a protein that serve serves as a loading control) in six
samples of HEK (human embryonic kidney) cells transfected with specific
plasmids .
We used different treatments: E2 and DEX (and a vehicle).
We can see from above that the picture is divided in two main parts: on the
left we have the results of GR and on the right we have the results of ER.
Each part shares three common lanes which represents the cells transfected
with an empty vector with different treatments (a vehicle, E2 and DEX).
The protein GAPDH is present in similar quantity in all the samples, meaning
that the loading was correct.
The levels of GR should have increased with DEX treatment, but the
treatment with DEX did not work well (very low expression in DEX lane on left
8 side of the figure). In contrast, the treatment E2 led to a slight increase in ER
levels (brighter band in E2 lane on right side).
4. Genotyping:
• Genotyping is the procedure used to determine differences in the
genetic make-up (genotype) of an individual by examining the
individual's DNA sequence using biological assays and comparing it to
another individual's sequence or a reference sequence.
• In our experiment we wanted to know the differences in the genotype
between wild type mice and mice with disrupted p23 gene. The mice
were result of a breeding between two heterozygous mice
(heterozygous means that they have one wild type and one knockout
copy of p23 gene). Knockout mice have an inserted cassette within p23
gene which results in the disruption of p23 gene and it is also the
reason why it is not possible to make p23 protein, as we could see in
lack of staining in IHC experiment.
During the experiment we used PCR (polymerase chain reaction),
which is a process used in molecular biology to amplify a single copy or
a few copies of a piece of DNA, generating multiple copies of a
particular DNA sequence.
• Results of the experiment:
mouse 1
2
3
4
5
6
Figure 8: Results of our genotyping experiment (PCR detecting the presence
or absence of p23 gene).
This photo shows different genotypes of six different mice.
We can see that there are six lanes, which represent the individual mice and
the lane on the right that contains the DNA marker that allows for
determination of the size of specific bands. The first lane shows just one band
9 that is around 830 bp (base pairs) in size, this means that the mouse has KO
genotype, the second is WT as the band that is present is 780 bp in size, the
third is KO, the fourth mouse has both WT and KO band so it is
heterozygous, the fifth is WT and the last one is heterozygous.
Conclusions:
According to the experiments we performed during study week, our aim
was to visualize gene and protein expression and we used variety of
techniques to do it .
It has been possible to understand, for example, that Estradiol activates
better ER than GR, or that the p23 knockout mice do not express the
protein p23.
To conclude, scientists are able to study and to visualize gene
expression in cells or tissues by virtue of these complex, but
fundamental techniques of biology, and this is also the reason why the
number of scientific discoveries is growing faster year after year.
References:
www.ncbi.nlm.nih.gov
www.piercenet.com
www.leinco.com
www.ptglab.com
Acknowledgements and affiliations:
To conclude, I want to thank all the staff of the Laboratory (my tutor Marta,
Melissa, Marco, Lila, Abhinav, Kaushik, Pablo) and also Prof. Didier Picard
for the patience that they had with me and for all the interesting information
that they explained and illustrated to me during the week of study.
For me, working in a Lab has been an amazing experience. I thank Swiss
Youth Science for having given me this wonderful opportunity.
10