Many studies, from reporter assays to protein localization to BRET and FRET, require successful transfection first. Yet, transfection can be tricky and difficult. There are many considerations when planning transfection of your cells including reagent selection, stable or transient experiment, type of molecule and endpoint assay used. Here we discuss these considerations to help you plan a successful transfection scheme for your experimental system.
With so many different methods of gene transfer, how do you choose the right transfection reagent or technique for your needs? For example, one reagent may work well with HEK-293 cells, but a second reagent is a better choice when using HepG2 cells. Promega offers the FuGENE® HD Protocol Database to help identify a protocol for your cell line when using the FuGENE® HD Transfection Reagent. A drop-down menu allows you to search the database by cell line, plate type and number of cells to be transfected. The Transfection Assistant also offers a drop-down menu to select cell lines and either FuGENE® HD or FuGENE® 6 Transfection Reagent for transfection conditions. The conditions should be considered only guidelines since you may need to optimize the transfection conditions for your specific application.
Transient Expression versus Stable Transfection
Another parameter to consider is the time frame of the experiment you wish to conduct. Is it short- or long-term? For instance, determining which promoter deletion constructs can still function as a promoter can be accomplished with a transient transfection experiment, while establishing stable expression of an exogeneously introduced gene construct will require a longer term experiment.
Cells are typically harvested 24–72 hours post-transfection for studies designed to analyze transient expression of transfected genes. The optimal time interval depends on the cell type, research goals and specific expression characteristics of the transferred gene. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The method used for cell harvest will depend on the end product assayed. For example, expression of the firefly luciferase gene in the pGL4.10[luc2] Vector (Cat.# E6651) is generally assayed 24–48 hours post-transfection, whereas the pGL4.12[luc2CP] Vector (Cat.# E6671) with its protein degradation sequences can be assayed in a shorter time frame (e.g., 3–12 hours), depending on the research goals and the time it takes for the reporter gene to reach steady state.
When performing a transient transfection, you can choose between a standard or reverse transfection protocol. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and assayed on day 2 or 3. Because the cells are added directly to the DNA, this process reduces the experimental time by one day and allows for high-throughput transfection of DNA in a plate- or microarray-format. For more information on reverse transfection including a protocol, read the PubHub article on the subject.
The goal of stable, long-term transfection is to isolate and propagate individual clones containing transfected DNA that has integrated into the cellular genome. Distinguishing nontransfected cells from those that have taken up exogenous DNA involves selective screening. This screening can be accomplished by drug selection when an appropriate drug-resistance marker is included in the transfected DNA. Alternatively, morphological transformation can be used as a selectable trait in certain cases. For example, bovine papilloma virus vectors produce a morphological change in transfected mouse CI127 cells.
Before using a particular drug for selection purposes, you will need to determine the amount of drug necessary to kill untransfected cells. This may vary greatly among cell types. Consult Current Protocols in Molecular Biology for additional information about designing experiments to test various drug concentrations and determine the amount needed to select resistant clones (i.e., generate a kill curve).
When drug selection is used, cells are maintained in nonselective medium for 1–2 days post-transfection, then replated in selective medium containing the drug. The use of selective medium is continued for 2–3 weeks, with frequent changes of medium to eliminate dead cells and debris, until distinct colonies can be visualized. Individual colonies can be isolated by cloning cylinders, selected and transferred to multiwell plates for further propagation in the presence of selective medium. Individual cells that survive the drug treatment expand into clonal groups that can be individually propagated and characterized.
Several different drug-selection markers are commonly used for long-term transfection studies. For example, cells transfected with recombinant vectors containing the bacterial gene for neomycin phosphotransferase can be selected for stable transformation in the presence of the neomycin analog G-418. Similarly, expression of the hygromycin B phosphotransferase gene from the transfected vector will confer resistance to the drug hygromycin B.
An alternative strategy is to use a vector carrying an essential gene that is defective in a given cell line. For example, CHO cells deficient in dihydrofolate reductase (DHFR) gene expression do not survive without added nucleosides. However, these cells, when stably transfected with DNA expressing the DHFR gene, will synthesize the required nucleosides and survive. An additional advantage of using DHFR as a marker is that gene amplification of DHFR and associated transfected DNA occurs when cells are exposed to increasing doses of methotrexate, resulting in multiple copies of the plasmid in the transfected cell.
Type of Molecule Transfected
Plasmid DNA is most commonly transfected into cells, but other macromolecules can be transferred as well. For example, short interfering RNA, oligonucleotides, RNA and even proteins have been successfully introduced into cells via transfection methods. However, conditions that work for plasmid DNA transfer will likely need to be optimized when using other macromolecules. In all cases, the agent transfected needs to be of high quality and relatively pure. Nucleic acids need to be free of proteins, other contaminating nucleic acids and chemicals (e.g., salts from oligo synthesis). Protein should be pure and in a solvent that is not detrimental to cell health.
Assay for Transfection
After cells are transfected, how will you determine success? Plasmids containing reporter genes can be used to easily monitor transfection efficiencies and expression levels in the cells. An ideal reporter gene product is one that is unique to the cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally, reporter gene assays are performed 1–3 days after transfection; the optimal time should be determined empirically. A direct test for the protein of interest, such as an enzymatic assay, may be another method to assess transfection success.
In the case of siRNA, success may be measured using a reporter gene or assaying mRNA (e.g., RT-PCR) or protein target levels (e.g., Western blotting).
If multiple assays will be performed, make sure the techniques you choose are compatible with all assay chemistries. For example, if lysates are made from transfected cells, the lysis buffer used ideally would be compatible with all subsequent assays. In addition, if cells are needed for propagation after assessment, make sure to retain some viable cells for passage after the assay.
For more information on transfection, including optimization and factors that influence efficiency, consult the Transfection chapter of the Protocols and Applications Guide.