Determination of the Selection Capacity of Antibiotics for Gene Selection

Abstract

Choosing a potent selection antibiotic (SA), is a crucial success factor when creating stably transfected cell lines using an antibiotic selection marker. The selection capacity of this antibiotic is defined as its ability to kill sensitive, untransfected parental cells, while leaving resistant, transfected cells unharmed. Currently, no procedure has been described to determine this selection capacity. Therefore, a protocol to obtain a numerical value, called the "selectivity factor" (SF), that defines the selection capacity of SAs is developed. The SF is determined by using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide) assay for both sensitive and resistant cells, and applies to commonly used cell lines. To prove the concept, the SF of the SA G418 and hygromycin B (HmB) on several cell lines is determined. The SF of G418 on BHK-21 cells is very high, indicating that G418 is an ideal SA for transfected BHK-21 cells. For HeLa cells, the SF of G418 is very low suggesting G418 is not an optimal SA for selecting transfected HeLa cells. For these cells, HmB would be a better choice. These conclusions are confirmed by an independent cell death assay. The SF identifies the most optimal SA for a certain cell line, reduces the risk of selecting spontaneously resistant cell clones, and streamlines the process of generating stable cell lines. Most importantly, the method is especially time saving when obtaining stable cell lines expressing toxic genes, and reduces culture times for generating large numbers of cell lines from the same parental cell line.

Development of a Cell-Based Biosensor for Residual Detergent Detection in Decellularized Scaffolds

“…The complete cell culture medium was replaced with a selected medium containing zeocin as a selective antibiotic, 24 h after transfection. The kill curve assay was used to determine the optimal zeocin dose, and 100 and 450 μg/mL of antibiotic as lethal concentrations for HEK293 and HDF cells, respectively, were chosen after 1 week of exposure for further experiments. , …”

“…The kill curve assay was used to determine the optimal zeocin dose, and 100 and 450 μg/mL of antibiotic as lethal concentrations for HEK293 and HDF cells, respectively, were chosen after 1 week of exposure for further experiments. 16,17 To perform single clone selection, transfected and reproduced cells were subcultured and highly diluted (50 cells per well on a 12-well plate). Using trypsin-soaked paper disks (Whatman filter paper; sterilized by autoclaving), clones were isolated and reproduced.…”

Transfection types, methods and strategies: a technical review

“…Linearized DNA, however, is more recombinogenic and can thus be more readily integrated into the host genome to achieve stable transfection (Von Groll et al, 2006;Hardee et al, 2017). The use of a plasmid vector does not guarantee constitutive transgene expression nor stable foreign DNA integration into the host genome (Delrue et al, 2018). In other words, as cells divide, a foreign gene will not be constitutively expressed and eventually lost if it has not been integrated into the host cell's genome (Mali, 2013).…”

Peer Review #1 of "Transfection types, methods and strategies: a technical review (v0.1)"

“…The use of a plasmid vector does not guarantee constitutive transgene expression nor stable foreign DNA integration into the host genome (Delrue et al 2018). In other words, as cells divide, a foreign gene will not be constitutively expressed and eventually lost if it has not been integrated into the host cell's genome (Mali 2013).…”