Applications:

The generation of stably-transfected cell lines is essential for a wide range of applications:

•  Cell line can be used for gene function studies

•  Drug discovery assays or the production of recombinant proteins can be carried out by cell lines.

•  In contrast to transient expression, stable expression of cell line allows long term, as well as defined and reproducible expression of the gene of interest.

Table 3: Types of culture system for cell lines

In a batch culture system, a mixed population of drug resistant cells is selected on plates or in flasks and can be used directly for experimental analysis. During a limiting dilution procedure, cells are usually diluted and selected e.g., in a 96-well plate for outgrowth of cell clones or single colony growth. Subsequently, colonies can be picked and used to generate monoclonal cell lines.

Culture conditions for generation of stable cell lines:

As for transient transfection experiments, culture conditions (passage number, split rhythm, etc.) of selected cell type are very important for the generation of stably-transfected cell lines. The American Type Culture Collection (ATCC®; www.atcc.org ) is a reliable source for various cell types. Generally, the cell line should be passaged two days before the experiment to promote good proliferation and cell physiology. Cell passage should not be higher than 30. Interference of higher passage numbers with integration efficiency is possible and may be cell-type dependent. Depending on the scope of experiment, cells can be cultivated as polyclonal batches or monoclonal single cell clones post transfection.

Transfection Method: Stable expression can be influenced by the transfection method used. The choice of transfection method determines which cell type can be targeted for stable integration. While biochemical transfection reagents (e.g., HiFect® Transfection Reagent) can be used to transfer DNA into standard cell lines, efficient delivery of DNA into notoriously difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods or Nucleofection. Unfortunately, viral methods suffer from several limitations, such as time consuming production of vectors and safety concerns.