Background
Cell transfection involves, rather trivially, cells. Although it may seem as though transfection is a generalized concept, each specific application of the technology requires a careful attention to the cell line being used and the kind of transfection to be achieved. Different cell lines also differ in the kind of conditions necessary for proper cell growth and successful transfection; a stem cell differs from epithelial cells in terms of the optimized conditions of growth and resistance to transfection. As a consequence of the differences among various cell types, different transfection setups and reagents should be used to optimize transfection efficiency.
Optimal Transfection Conditions
Researchers have a plethora of options when it comes to selecting an optimal transfection reagent to facilitate cellular uptake of nucleic acids (such as proteins, siRNA, DNA and miRNA). In addition to general purpose transfection reagents, many cell line specific reagents are also commercially available. As the name indicates, cell line specific transfection reagents are pre-optimized for cell lines, which are established immortal cultures of a specific cell type.
The basic objective of transfection experiments is to study cellular processes that trigger protein synthesis. This can be accomplished in a transient or stable transfection. The gathered data can be used for various purposes, from gene therapy to plant and livestock production. To reach end goals quickly and efficiently, scientists need to set up optimal transfection reactions every time.
Cell lines thrive well and propagate when grown in favorable living conditions. Examples of cell lines include hamster ovarian cells (CHO), human pancreatic cells (AsPC-1), human neuroblastoma cells (CLBPEC), human umbilical vein endothelial cells (HUVEC), cord blood stem cells, human ovarian cancer cells (SKOV3), human hepatoma cells (HUH7) and many others.
Because inherent properties of differing cell lines can impact the success of any transfection, choosing the right method of transfection alongside the reagents is also important. There are physical methods (electroporation), chemical methods (calcium phosphate), viral methods, and more to choose from. Cells like primary cells may experience better transfection with electroporation, while other cells may work better with liposome-mediated transfection.
Optimizing transfection efficiency involves taking into consideration multiple factors: amount of transfected nucleic acid, presence/absence of serum, charge ratio of reagent to DNA (if applicable), and how long the cells are exposed to the reagent. Using reporter genes can help determine what the optimal conditions are for each cell type.
Transfection Reagent Advantages
Using the appropriate transfection reagent increases the efficiency of transfection and limits the amount of cytotoxicity. There are several pre-optimized transfection reagents available on the commercial market. Cell line specific transfection reagents come with protocols to assist researchers in establishing efficient transfections with minimal toxicity.
Data reveals that the cell membrane phospholipid bilayer plays an important role in restricting exogenous DNA or RNA molecules from entering the cell. Successful transfection depends on several factors, such as preferred reagents, the protocol used, type of cells to be transfected and desired outcome of the transfection process. Some cells are inherently difficult to transfect, such as primary cells or suspension cells. Some cell types can exhibit better transfection efficiency when transfected in a certain way, such as reverse transfection versus forward transfection. Off-target effects and cytotoxicity of the transfection system often leads to low transfection efficiency.
Cell lines may undergo significant changes as the culture matures, confluency increases and nutritional medium becomes scarce. As a result, the efficiency at which they can be transfected varies. Scientists have observed that cells may show poor transfection results if they undergo the process soon after being thawed from cryopreservation. The same cells may exhibit better transfection efficiency after recovering completely from the effects of cryopreservation. Good laboratory practice is to use cell lines purchased from authorized sources, such as DSMZ and ATCC, and to optimize the cell line growth prior to starting any transfection process. In addition, a number of transfection services are commercially available from several companies.
Many protocols are available and already optimized to achieve the most efficient, viable and reproducible performance across multiple cell types. If these protocols do not work, cell-specific protocols may be a better choice to optimize your experiment. CROs such as Altogen Labs employ experienced scientists who can recommend optimal transfection reagents and procedures for different cell lines.
Different transfection reagents, whether general purpose or cell line specific, work differently to insert foreign DNA, siRNA or protein into cultured cells. While some reagents work by shrinking the nucleic acid to be transfected, others are particularly effective in the serum contained in cell cultures. Certain transfection reagents have been designed with positively charged molecular linkers such that external nucleic acids readily coil around these molecular arms and then enter cells when the reagent molecules are endocytosed.