Biotechnology Letters, Vol.43, No.5, 949-958, 2021
Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry
Objective Chromovert (R) Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression. Methods The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes. Results Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (alpha beta gamma-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-alpha beta 1 beta 2), four unique gamma-aminobutyric acid A (GABA(A)) receptor ion channel subunit combinations alpha 1 beta 3 gamma 2s, alpha 2 beta 3 gamma 2s, alpha 3 beta 3 gamma 2s and alpha 5 beta 3 gamma 2s, cystic fibrosis conductance regulator (CFTR), CFTR-Delta 508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3 ' untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes. Conclusions Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.