Generating stably transfected cells begins with a transient transfection, followed by an infrequent but important and serendipitous process. In a small proportion of transfected cells, the foreign gene is integrated into the cells’ genome. The hallmark of stably transfected cells is that the foreign gene becomes part of the genome and is therefore replicated. Descendants of these transfected cells, therefore, will also express the new gene, resulting in a stably transfected cell line.

When developing a stable transfection, researchers use selectable markers to distinguish transient from stable transfections. Co-expressing the marker with the gene of interest enables researchers to identify and select for cells that have the new gene integrated into their genome while also selecting against the transiently transfected cells. For example, a common selection method is to co-transfect the new gene with another gene for antibiotic resistance (such as the neomycin resistance gene, neo) and then treat the transiently transfected cells with the appropriate antibiotic for selection (such as geneticin or G418 for neo-transfected cells). Only the stably transfected cells with resistance to the antibiotic will survive in long-term cultures, allowing for the selection and expansion of the desired cells.

Stable Expression Cell Line Construction

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