Generating stable cell lines is a common practice in molecular biology research that involves integrating a foreign gene of interest into the genome of a host cell line. Here are the general steps involved in generating stable cell lines:
- Selection of host cell line: Select a host cell line that is suitable for the research question and has the ability to be genetically modified. Common cell lines used for stable cell line generation include HEK293, CHO, and HeLa cells.
- Design and construction of expression vector: Design and construct a suitable expression vector that contains the gene of interest along with a selectable marker, such as a neomycin resistance gene or puromycin resistance gene.
- Transfection: Transfect the host cells with the expression vector using a suitable method, such as electroporation or lipofection. The cells that take up the expression vector will then express the gene of interest along with the selectable marker.
- Selection and screening: Add a selective agent, such as neomycin or puromycin, to the culture media to kill off any untransfected cells. The surviving cells are then screened for the gene of interest expression using methods such as PCR or western blot.
- Isolation of clonal cell lines: Once the expression of the gene of interest is confirmed, isolate clonal cell lines by limiting dilution or by using a fluorescence-activated cell sorter (FACS) to obtain homogeneous cell populations.
- Confirmation of stable expression: Confirm that the gene of interest is stably integrated into the genome of the clonal cell lines by analyzing the expression of the gene over multiple passages.
Generating stable cell lines can be a time-consuming process, but it provides researchers with a consistent source of cells that express the gene of interest at a predictable level. These cell lines can be used for various applications such as drug screening, disease modeling, and functional analysis of the gene of interest.