Transfection and Reporter Gene Assays
Molecular Biology Lab #6
Background:
Transfection is the process of introducing naked DNA molecules into cells. It is one of
the most frequently used techniques in molecular biology. Transfection of cells is usually
accomplished in one of three basic methods. Chemical methods of transfection include
calcium phosphate or lipofection. Physical methods include electroporation or the “gene
gun”. In addition, gene transfer can also be mediated with high efficiency by viruses such
as adenovirus or retroviruses.
Transfection can be categorized into 2 major types, stable and transient. Transient
transfection is temporary and high level expression of foreign genes. Expression lasts for
several days, but is lost as the DNA never integrates stably into the host cell DNA. In
contrast, stable transfection occurs with a lower frequency (10 to 100-fold lower), but
expression is maintained for the long term because the foreign DNA does integrate into
the host DNA.
Cotransfection is transfection of cells with 2 different DNAs at the same time. In the case
of transient transfection, it is often useful to cotransfect cells with a reporter gene, such as
-galactosidase or green fluorescent protein as an indicator. All cells that take up and
express the DNA turn color and can be counted. This allows one to determine the
efficiency of transfection. In the case of stable transfection, cotransfection is often used to
introduce a selectable marker (such as an antibiotic resistance gene). Since only one in
1000 cells might be stably transfected, it is necessary to select these cells from the total
population. Cells that express the selectable marker also take up and express the other
gene of interest. Selectable markers that are commonly used include:
1. The neomycin resistance gene (neo): the most common method because it works
so well. It is a bacterial gene that converts the antibiotic G418 (which blocks
protein synthesis) to an inactive form.
2. Thymidine kinase: the oldest method. When cells are grown in the presence of
aminopterin, only cells that express the TK gene can survive.
3. Hygromycin B phosphotransferase: a bacterial gene that inactivates the antibiotic
hygromycin B.
4. puromycin-n-acetyl transferase: an enzyme that acetylates and inactivates the
antibiotic puromycin.
There are several different methods that are frequently used for transfection, including
lipofection, calcium phosphate, electroporation, and bolistics. Lipofection is a procedure
in which the DNA is complexed within lipid droplets. The droplets interact directly with
the cell membrane and fuse. The DNA is liberated into the cytoplasm and some
eventually reaches the nucleus. Lipofection is one of the most efficient methods of
transfection. However, it is also relatively expensive, it can be toxic to cells, and it cannot
be used with cells growing in serum. Lipofection, like other forms of transfection, works
much more efficiently if the cells are rapidly growing. This is because the nuclear
membrane is absent during cell division, allowing easier access to the host DNA.
Calcium phosphate is another popular method for transfection. In this procedure, the
DNA is precipitated with calcium phosphate aggregates. The cells phagocytize the
aggregates and the DNA is released into the cytoplasm and eventually reaches the
nucleus. This is the oldest method and its main advantage is that it is cheap and easy to
perform. However, calcium phosphate transfection is not as efficient as lipofection and
the precipitates often cause cytotoxicity.
Electroporation is another method of transfection in which cells are exposed to an electric
shock. This induces transient aqueous channels in the membrane for DNA to enter the
cytoplasm. On the positive side, electroporation is rapid and simple, and it works on
almost all types of cells. However, one needs special equipment such as an electroporator
to shock the cells. The transfected cells also have high cytotoxicity after shocking.
An interesting method of transfection was developed for plant cells that have a thick cell
wall. Bolistics or the ‘gene gun’ is used to directly blast the DNA into the cell. Usually
the DNA is attached to small particles of gold or tungsten. This method works especially
well in plant cells. It is also useful for introducing DNA through the skin of live animals
(vaccination). On the negative side, it is expensive. You need a special gun and expensive
ammo.
Gene transfer can be performed very efficiently using viral vectors, either adenoviruses
or retroviruses. Adenoviruses are small DNA viruses that often produce flu-like
symptoms in humans. The adenovirus genome can be altered by removing genes for
virulence and substituting an exogenous cloned gene of interest. When the target cell is
infected, the adenovirus DNA enters the cytoplasm and the foreign gene is expressed.
However, the virus cannot replicate and kill the cell because its virulence genes are
missing. Adenovirus infection has been used for gene therapy in humans, although
experiments are in the very early stages. The advantage of the adenovirus method is that
it is very efficient. Almost all cells can be infected. The DNA does not integrate into the
host cell genome, so there is little chance of mutagenesis and cancer. Another advantage
is that adenoviruses infect cells regardless of whether they are dividing or stationary.
However, since the DNA fails to integrate, the treatment must be repeated frequently
(every few weeks, as expression is lost).
Retroviruses are small RNA viruses that can cause cancer in animals. Retrovirus vectors
are constructed similarly to the adenovirus vectors. A small piece of he retrovirus genome
is removed and the exogenous gene of interest is cloned into the virus DNA. When cells
are infected with the recombinant retrovirus vector, the DNA enters the cytoplasm and
heads for the nucleus. If it integrates, the retroviral vector is expressed stably. This is an
advantage for long-term expression. However, retroviruses only infect rapidly growing
cells and most cells in the body are not rapidly dividing. In addition, integration into the
host genome can lead to DNA rearrangments and mutations, which are associated with
cancer.
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Transfection is often used for setting up reporter gene assays. A reporter gene is any gene
that ‘reports’ where it is expressed and how much it is expressed. Its activity can be
measured quantitatively. A reporter gene assay is developed by cloning the reporter gene
into a cellular gene in place of the normal coding region. The reporter gene is now
regulated by the normal promoter and enhancer elements that control expression of the
original gene. When this recombinant reporter gene is transfected into cells, the normal
regulatory regions induce expression of the reporter gene, and the level of expression can
be easily measured in a quantitative manner. For example, a reporter gene might be
cloned into the coding region of the cystic fibrosis gene. This would allow a researcher to
transfect the gene into cells and treat with different drugs. It would be possible to
determine the effects of these drugs on expression of the gene. Since reporter gene
products are easy to measure, this method is easier and more reliable that trying to
measure the actual product of the cystic fibrosis gene.
Reporter gene assays are used for two major purposes. First, as mentioned above, they
are a valuable tool to measure how drugs or other genes might alter the level of gene
expression. For example, the reporter gene might be cotransfected with another gene of
interest and the transfected cells could be analyzed for how one gene product influenced
expression of the other (the reporter gene). Second, reporter gene assays are useful for
determining the percentage of cells that actually take up and express the transfected
DNA. When cells are transfected with DNA, some cell types take up and express DNA
more efficiently than others. If a researcher wanted to compare the biological effects of
one gene in two different cell types, the procedure would be to cotransfect both cell types
with the reporter gene and the gene of interest. This would allow one to control for
differences in the ability of the two cells to take up an express DNA (if one cell only took
up half as much DNA, one would expect that they would also exhibit a proportionally
lower response to the other cotransfected gene.
What are some common examples of reporter genes? The LacZ gene of E. coli encodes
the -galatosidase enzyme (-gal). The normal function of this protein is to cleave
galactopyranosides into galactose and glucose. However, it also cleaves the artificial
substrate, X-gal, into an insoluble bright blue reaction product. This allows one to
determine which cells took up and expressed the DNA because they are bright blue. The
-gal assay is easy and sensitive. This assay will be performed in lab.
Green fluorescent protein (GFP) is another important reporter gene. It was isolated from
bioluminescent jellyfish. It is a small (26 kD) stable protein that emits light at 510 nm. It
is frequently used to determine the percentage of cells that take up and express DNA.
GFP is also useful in gene therapy. It can be introduced into cells that will be transferred
to patients, and it allows researchers to follow the cells throughout the body and
determine their survival. It is also possible to create a fusion protein between GFP and
other cellular proteins. These proteins become tagged with a green marker, which allows
one to follow their movements within the cell during protein synthesis and processing.
The advantage of GFP is that it requires no substrate, it constantly emits green light. The
drawback is that the emission is not particularly strong. Therefore one must use a very
strong promoter to increase expression of GFP so that one can see it within the cell.
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Luciferase is another useful reporter gene. Luciferase is the protein that makes fireflies
glow in the dark. It is found only in the gut of fireflies and several other marine
organisms. The reaction is:
ATP + luciferin sustrate + 02 → AMP + oxyluciferin + LIGHT
Natural luciferase can cause cells to become sick because it is targeted to and collects at
high levels within the peroxisomes, a subcellular organelle. The luciferase gene has been
engineered by several biotech companies to remove the peroxisome targeting sequence,
and this reduces the toxicity. The great advantage of using luciferase as a reporter gene is
that it is extremely sensitive. There is no background because luciferase is not expressed
in mammalian cells. However, luciferase does require the luciferin substrate for activity.
It also requires expensive equipment such as a luminometer or scintillation counter to
detect the signal.
The oldest reporter assay relies upon the CAT gene (chloramphenicol acetyl transferase).
CAT covalently modifies chloramphenicol by adding an acetyl group. Acetylated
products can be detected using chromatography followed by autoradiography. The
advantage for using the CAT gene is that it is not expressed in mammalian cells. Thus,
there is no background. However, the CAT gene is not as sensitive as the luciferase
assay. It is also rather expensive, time consuming, and you need to use radioactive
isotopes.
Objective:
The objective of this lab is to learn about different methods of transfection and to identify
the strengths and drawbacks of each. Students will perform transfection using the
lipofection method. They will introduce 2 reporter genes (-gal and GFP) into cultured
cells, and perform assays to detect expression and count the percentage of transfected
cells. Students will also introduce a selectable marker (neo) into cells and perform
selection of stable transformants in the presence of G418.
Materials:
Lipofectin transfection kit (Life Technologies)
Cells growing in 60 mm dishes
Eppendorf tubes
Yellow tips and micropipetters
Reporter genes (-gal and enhanced GFP from Clontech Corp.)
KSF-M cell culture medium with and without growth factors
-gal detection kit (Invitrogen Corp.)
Fluorescent microscope
G418 solution at 200 g/ml in KSF-M
Cell culture facility and supplies
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Methods:
1. The first lab period will involve transfection of cells in 60 mm dishes with 3
recombinant plasmid DNAs. These include -gal, enhanced GFP, and neo.
1.
Place 2 g of plasmid DNA in 1.5 ml eppendorf tube. The number of tubes
needed (6) will be twice the number of different types of DNA that you will
be transfecting. Separate the tubes into pairs, as they will later be mixed
together.
2.
Remove the growth media from the plates of cells that are to be transfected
and add 2 ml of basal media.
3.
In one of the two eppendorf tubes, dilute the desired amount of DNA with 250
μl of basal media (2 g divided by the DNA concentration in g/ml). After
dilution of DNA, add 8 μl of PLUS reagent. Mix the tube by inverting a few
times and incubate at room temperature for 15 minutes.
4.
In the second eppenorf tube place 250 μl of basal media and 12 μl of
lipofectAMINE reagent. Mix tube by inverting it a few times and incubate at
room temperature for 15 minutes.
5.
After both tubes have incubated for 15 minutes, add the contents of the second
eppendorf tube to the first tube and mix by inverting a few times. Allow the
mixture to incubate for an additional15 minutes at room temperature. This
allows the liposomes to form.
6.
Add 0.5 ml of the transfection mixture to each 60 mm culture dish and let the
transfection mixture remain on cells for 3 hours at 37˚C.
7.
After the 3 hours, remove the transfection mixture and wash once with PBS.
Then, feed the dishes with 5 ml of normal KSF-M.
8.
Feed with fresh KSF-M the next day and observe cells for signs of toxicity.
2. The second lab will involve detection of transient transfection efficiency by measuring
the percent of cells that stain for -gal and GFP. You will use the Invitrogen -gal
staining kit. Each student will also start selection of stably transfected cells by treating
the neo-transfected cultures with the antibiotic G418. Do not treat the GFP or -gal
transfected cultures with G418!
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1. Dilute the 10x PBS and 10x fixative solutions with distilled water to make 1x
solutions. You will need to make up 10.5 ml of 1x PBS and 3 ml of 1x fixative
solution per 60 mm dish.
2. The following procedure is for staining 1 x 60 mm plate. Remove the growth
medium from the transfected cells and rinse the dish once with 2.5 ml of 1x PBS.
3. Fix the cells with 3 ml of 1x fixative solution for 10 minutes at room temp.
4. While the cells are fixing, prepare the staining solution. Be sure to use
polypropylene plastic (15 ml conical tubes with orange tops). Add 25 l of
solution A, 25 l of solution B, 25 l of solution C, 125 l of X-gal substrate
solution, and 2.3 ml of 1x PBS. Mix well.
5. Rinse the plate with 2.5 ml of 1x PBS twice.
6. Add 2.5 ml of staining solution to the plate. Incubate at 37C for 0.5 to 2 hours, or
longer until the cells stain blue. Rock the plates occasionally to ensure even
coverage of the plate. Note: if cell confluence is high, do not stain plates more
that 2 hours as the stain will travel from cell to cell via gap junctions.
7. Check the cells under the microscope (x200 total magnification) for the
development of blue color. Count the total cells and blue cells in 5 random fields
of view and use the average to estimate transfection efficiency.
8. Calculate the percentage of cells expressing -galactosidase using the following
formula:
Total number of blue cells x 100 / total number of cells = transfection efficiency
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