Stable cell lines for assays are widely used in research, as tools to discover new pathways, or for discovery of therapeutics. Development of cell based assays requires a single-clone stable cell line that has been validated for expression of the gene of interest. GenScript is capable of developing recombinant stable cell lines with most difficult-to-transfect cells and high expression of difficult-to-express genes using our lentiviral-based platform, CellPower™. GenScript can develop stable cell lines using non-lentiviral approaches as well. Below are some applications-based case studies, showcasing stable cell lines developed by GenScript.

Case study: Generation of an apoptosis-resistant cell line to improve screening against genotoxicity

Genotoxic screening for aneugens and clastogens can be assayed by micronucleus detection via flow cytometry. However, this type of assay often produces false positives due to the presence of apoptotic bodies caused by cytotoxicity, not genotoxicity.

To overcome false positives, scientists from a major pharmacetical company developed a more robust assay by implementing an apoptosis-resistant stable cell line (BCL-xL overexpression in TK6 cells) in the FACS-based micronucleus assay. Additionally, presence of aneugen marker, phosphorylated histone-3 (H3) and double strand breakage marker, γH2AXser139 (γH2AXDSB) was assessed to further validate micronucleus-inducing compounds.

GenScript's CellPower™ lentiviral-based stable cell line service was able to deliver the critical recombinant stable cell line for development of this highly specific genotoxic assay.

Generation of the apoptosis-resistant stable cell line

Generation of the apoptosis-resistant stable cell line

qPCR and Western blot validation of TK6 Bcl-xL single stable clones

qPCR results

qPCR analysis shows high mRNA expression of BCL-xL in stable clones, compared to un-transfected control

western blot results

Protein expression of BCL-xL in single stable cells confirmed via western blot

Clastogen (Etoposide) Aneugen (Vinblastine) Cytotoxicant (Tunicamycin)
Micronucleus / toxicity profile Clastogen - toxicity profile
Clastogen - toxicity profile
Aneugen - toxicity profile
Aneugen - toxicity profile
Cytotoxicant - toxicity profile
Cytotoxicant - toxicity profile
H3-P (Aneugen marker) profile Clastogen - H3-P profile
Clastogen - H3-P profile
Aneugen - H3-P profile
Aneugen - H3-P profile
Cytotoxicant - H3-P profile
Cytotoxicant - H3-P profile

The above figures show that there are similar levels of cell growth and toxicity between both the wild type and BCL-xL overexpressing TK6 cell lines. Also, TK6-BCL-xL exhibited fewer apoptotic bodies compared to wildtype. The BCL-xL cell line produced a positive response to clastogens (Etoposide) and aneugens (Vinblastine), but not cytotoxicants (Tunicamycin). Additionally, clastogens demonstrated elevated levels of γH2aXDSB, while aneugens demonstrated elevated levels of H3-P and cytotoxicants demonstrated no elevation of either γH2aXDSB or H3-P.

Richard A. Spellman, et al., "Strategies to Avoid Misleading Positives and to Identify Genotoxic Mechanisms in the Flow Cytometric In Vitro Micronucleus Assay in TK6 Cells" (poster presentation, 2014 Annual Genetic Toxicology Association Meeting, Newark, DE, May 7-8, 2014), accessed September 4, 2014 http://www.gta-us.org/scimtgs/2014Meeting/posters2014.html#poster1

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