Generation of Stable Cell Lines

Stable cell lines are used in many applications, including protein production, drug screening, and functional studies. The generation of stable cell lines involves the introduction of a foreign gene of interest into the host cell genome and the selection and expansion of cells that stably express the gene. Here is a general overview of the steps involved in generating stable cell lines:

  1. Vector selection: The gene of interest is cloned into a vector that can be used for transfection. The vector should have a selectable marker, such as neomycin resistance or hygromycin resistance, to enable selection of cells that have incorporated the vector.
  2. Transfection: The vector is introduced into the host cells using various methods, including chemical transfection, electroporation, or viral transduction.
  3. Selection: Cells that have incorporated the vector are selected using a selectable marker, typically a drug that kills cells that have not incorporated the marker. The concentration of the drug is optimized to select for cells that have stably incorporated the vector.
  4. Clonal isolation: Individual cells are isolated and expanded to generate clonal cell lines that stably express the gene of interest.
  5. Screening: Clonal cell lines are screened for expression of the gene of interest using various methods, including Western blotting, immunocytochemistry, and RT-PCR.
  6. Validation: Stable cell lines are validated to ensure that they retain the desired phenotype and function. This can include functional assays, such as protein production, drug screening, or cell-based assays.
  7. Maintenance: Stable cell lines are maintained by regular subculturing and testing to ensure that they retain stable expression of the gene of interest.

Overall, the generation of stable cell lines is a complex process that requires careful vector selection, transfection, selection, clonal isolation, screening, and validation techniques to obtain pure and functional cell lines that stably express the gene of interest.