Engineering a mutation or overexpressing a recombinant protein to study and characterize its function in mammalian cells is no easy task. Luckily, Chinese hamster ovary (CHO) cells, which have been a mainstay in the lab since the 1950s, represent a relatively easy mammalian model system to engineer. There are several methods to choose choose from when trying to engineer your cell line(s) of interest. Bear in mind that some cells lines may be a bit more difficult to engineer than others. In this article, I will introduce a few of the most popular transfection methods used in cell line engineering today.
- Employs a liposome-based transfection reagent that is taken up by cells when mixed with the genetic material (e.g., plasmid, siRNA).
- Easy to use and doesn’t require many steps or specific expertise.
- Typically, you will need a transfection reagent and a compatible growth medium (usually low in serum and well buffered to keep your cells happy during the transfection process). Different vendors have their own formulated reagents, and some are even designed for specific cell lines so check to see if your cell line has its own optimized lipofection formula.
- Compatible with a range of cell types (e.g., HEK 293 and CHO cells).
Bear in mind that lipofection reagents can be pricey, but are usually worth it in the long run.
2) DEAE-Dextran (Diethylaminoethyl-Dextran)
- This is a cationic polymer that binds-negatively charged DNA and proteins, forming a structure that can then be endocytosed by cells.
- Important optimization parameters include polymer concentration, the ratio of nucleic acids or protein to polymer, and duration of transfection.
- Is most effective for transfecting adherent cells, but can be used for some suspension cell lines.
The downside of this method is that some cell types are particularly sensitive to DEAE-dextran, which may lead to morphological changes as well as inhibition of cell growth.
3) Calcium Phosphate Co-Precipitation
- DNA is mixed with a concentrated calcium chloride solution which is then added to a buffer containing phosphate ions (e.g., HEPES), to form a calcium phosphate-DNA precipitate.
- This precipitate is then added to and taken up by your cells.
- This is the cheapest method of transfection and is widely used.
Drawbacks include variability in transfection efficiency due to issues with the precipitate (e.g clumping of the DNA) and cytotoxicity in some cell types.
Non Chemical-Based Methods
- Cells experience short pulses of an electric field, essentially shocking them into taking up foreign genetic material. Electroporation is also suitable for transfecting proteins.
- A number of electroporation devices exist and no specific expertise is required for operation. Make sure to ask the supplier for a demo of any device to make sure it works for your specific cell line before you buy!
- Each electroporation apparatus applies an electric field to the cells, via the electroporator.
- The electroporator is set up to fit either a cuvette or a pipet tip, which contains an electrode to apply the voltage directly to the suspended cells.
- After the conditions are set and the cells are suspended in either the cuvette or the tip, you place the sample in the electroporation unit and click a button! Make sure that there are no bubbles present, as this may affect transfection efficiency.
- Add recovery media to cells immediately after electroporation for maximum transfection efficiency.
- Optimization of voltage, duration and number of pulses can increase the permeability of the foreign material, but you will need to carefully optimize these parameters for each cell line.
Bear in mind that not all cell types tolerate electroporation and it may cause some cell types to die.
2) Viral transduction
- This involves using viral vectors to transfer genetic material to primary mammalian cells, which are often difficult to transfect.
- May require considerable safety precautions (e.g. containment) due to the risk of viral transmission.
- You may need to use several vectors to reduce the risk of a recombination event.
- Requires packaging cell lines (e.g., HEK 293 T cells) to produce the viral particle that will carry your transfecting DNA.
Tips and Tricks for Successful Transfection
Transfection of mammalian cell lines is not trivial. Deciding which method works best for you depends on many factors such as:
- Your lab budget
- Access to equipment (i.e., electroporation device)
- Your cell type – immortalized or primary cell lines
- Whether you want to stably or transiently transfect cells (Principles and Mechanisms of Mammalian Cell Transfection)
- How well your cells can take up control plasmids
As I’ve shown you in this article, there are a number of methods to choose from when transfecting cells, and you can do yourself a big favor in the long run by comparing different reagents and protocols early on. Luckily, most of the equipment and consumables are available at fairly affordable prices. The best transfection method is usually the one that gives the best transfection efficiency. Hopefully the final tips below will leave you with excellent transfection efficiencies in no time!
1. Add a Selection Pressure!
Essentially, you can try to ‘force’ your cells to take up and retain foreign genetic material.
Example 1: Antibiotic Resistance
The genetic material you are trying to introduce (typically a plasmid) will bear an antibiotic-resistance-conferring gene. After the initial transfection, the cells might be a little ‘sickly’, needing time to recover, but after the cells a day or two, you can expose them to an antibiotic. Cells that have not taken up the plasmid will not be able to withstand the antibiotic and will die, so you can continue your work with only the transfected cells. This is a great screening tool!
Example 2: Dihydrofolate Deficiency
Some cell lines, like CHO-DG44 are dihydrofolate-deficient, and dihydrofolate is essential for 1 carbon transfer chemistry and therefore metabolism. You can bypass the requirement for this gene by supplementing 1-C sources in the medium, but once you take this away the cells will die. You can however get the cells to survive if the genetic material that you want the cells to take up also carries the Dhfr gene.
2. Determine Your Transfection Efficiencies Early
It is important to know how good your transfection method actually is. Knowing this can save you from trying to troubleshoot and optimize a protocol that might just never work!
One quick and easy way to compare different transfection methods is to use a plasmid carrying a fluorescent-tagged protein so that you can physically see the transfected cells and count how many fluoresce under a microscope. You will probably need to try several concentrations of DNA/siRNA to determine the optimal transfection efficiency for your cell line.
3. Try to Work with Immortalized Cell Lines
Immortalized cell lines are indisputably easier to work with than primary cell lines, but bear in mind that you may still observe cytotoxic effects and these will affect the overall transfection efficiency.
Do you have any transfection tips to share with us? Feel free to post them in the comments section.Image Credit: nick farnhill