Cell Line Generation

Transfection of Cell Lines

  • We determine the optimal seeding density (70% confluence) of your cell line of interest, and optimal antibiotic concentration (>90% cell death after 10 days) prior to transfection

  • For transfection, a delivered DNA construct is added to a cell line of interest in combination with Lipofectamine 3000 (optimized with different amounts of DNA and Lipofectamine). We then culture transfected cells in a flask in the presence of antibiotics to select transfected cell pools.

  • Knockdown and overexpression are validated either by immunoblotting or flow cytometry. Transfection efficiency determined via flow cytometry occurs via staining the cells with an antibody targeting the respective protein, either on the surface or intracellularly (figure 1)

  • The cell lines with the lowest or highest protein expression are chosen for subsequent experiments. Clones with similar expression levels can be generated via single cell cloning. Using fluorescent activated cell sorting, we can select for a cell population with a certain expression of a surface marker (figure 2)

Oncolines® - Cell Line Generation - Transaction of cell lines
Figure 1: Schematic overview of study in which transfected cell line is generated.
Oncolines® - Cell Line Generation - Cells transfected with expression vector
Figure 2: Cells with or without expression of the protein of interest will be positively or negatively charged, resp. Deflection plates ensure that the cells with a positive and negative charge are separated. In this example, cells with high expression of a certain receptor can be bulk- or single cell-sorted into tubes or a 96-well plate, resp.

Resistant cell lines

Drug resistance is a major problem in cancer therapy. Many aspects of clinical drug resistance can be studied in drug resistant cell lines generated in vitro. We can test whether your compound can overcome drug resistance as a single agent or in combination with another therapy. Moreover, the mechanisms underlying drug resistance can be studied in drug resistant cell lines.

We offer profiling of compounds in resistant cell lines available at Oncolines, or we can generate cancer cell lines that are resistant against your compound.

Resistant cell lines are generated by exposing cells in culture to gradually increasing doses of your drug of interest (figure 2). Drug resistance is confirmed in viability assays and cell lines can be characterized by DNA sequence analysis. Pathway analysis is performed on RNA sequencing data to determine whether signalling pathways in resistant cell lines are altered in comparison to the parental cell line. Immunoblot analysis is performed to validate expression of target proteins or to determine up- or downregulation of proteins in a specific pathway.

Oncolines® - Cell Line Generation - Resistant cell lines
Figure: Schematic overview of generation of resistant cell lines. Cells are cultured in presence of drug of interest. The dose of drug will be gradually increased overtime.

Information Needed?

For more information about Cell line Generation, please contact us