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Chromovert® Technology: Chromo-Tag™ Probes and Plasmids

CHROMOVERT TECHNOLOGY is a newly published research tool for rapid creation of stable cell lines. The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more genes.


Mammalian cell lines, especially those produced using immortalized lines like human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells, are widely used in biological research, including in drug discovery and for biologics production. In general, cell line production begins with the introduction of one or more plasmids encoding cDNAs of interest into a cell culture. The goal is to produce, in a reasonable time frame, clonal cell lines that meet desired criteria for an application of interest. Despite multiple advances in cell engineering, the rapid creation of robust and multi-gene cell lines remains of reach - until now.

Originating at The Rockefeller University, Chromovert Technology is a cell engineering tool for rapid production of stable cell lines expressing one or more genes. The method is based upon the broadly-applicable principles of fluorescence-resonance energy transfer (FRET) and nucleic acid hybridization using fluorogenic oligonucleotide signaling probes originally reported for in tubo qRT-PCR applications, transfected into living cells. The termini of the signaling probes are covalently linked to a fluorophore or quencher paired to absorb its emission. The termini are designed to form a 4-7 base-pair stem juxtaposing the fluorophore and quencher pair. In the presence of target sequence, hybridization of the sequence-specific probe results in a fluorogenic conformational change. Flow cytometry is then used to detect and isolate positive cells that fluoresce above background. Thousands of individual clones can then be isolated and expanded using automated cell culture methods. Functional testing over time in the absence of selective pressure is used to select final clones.

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a) cDNAs are subcloned for expression of mRNAs comprising 3' untranslated plasmid-encoded Chromo-Tag™ sequences for detection using fluorogenic oligonucleotide signaling probes. Protein expression products remain untagged.
b) To create cell lines, one or more Chromo-Tagged cDNAs are transfected into cells, the transfected cells are exposed to differentially-labeled signaling probes and individual positive cells are isolated using flow cytometry. Downstream testing is used to select final cell lines.

Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry. Shekdar, K., Langer, J., Venkatachalan, S. et al. Cell engineering method using luorogenic oligonucleotide signaling probes and flow cytometry. Biotechnol Lett (2021). https://doi.org/10.1007/s10529-021-03101-5

For more information and for complete pChromo-Plasmid™ sequences, go to Secondcell Bio, LLC at https://www.secondcellbio.com/.

Click here to order Chromo-Tag™ Probes and Plasmids.

Research materials, including fluorogenic probes and pChromo-Plasmids™ comprising Chromo-Tags™, are licensed for research purposes only. An individual license for commercial use may be obtained by contacting Secondcell Bio, LLC at cells@secondcellbio.com. Gene Link in collaboration with Secondcell Bio, LLC is making these products available for research use only without further licensing.
Chromovert® Technology is a registered trademark of Chromocell Corporation. Chromo-Tag™, pChromo™, pChromo-Plasmid™, Secondcell™ and Secondcell Bio™ are trademarks of Secondcell Bio, LLC.

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