Manipulation and isolation of cells using molecularbeacons and flow cytometry for multi-gene cell lineproduction

The drug discovery process, in which compounds are screened and evaluatedfor therapeutic use, has resulted in safe and effective therapies for a variety ofdiseases. However, assessments of new compounds for drug development arenotoriously long and costly. Removing these barriers using new gene-editingtechnologies like CRISPR is key to expanding drug discovery. But selectivecompounds for different combinations of heteromultimeric proteins andindividual members of large gene families are lacking. Yet even with CRISPRand other recent cell engineering innovations, the production of just one stablemultigene cell line remains a tedious and unpredictable process.Also, CRISPR-Cas۹ specifically cleaves DNA to give the mutant sequence inits genome during the repair process. This method can be toxic to cells,because the enzyme Cas۹, a molecular "scissors" for cutting DNA strands,also cuts non-target parts. But CHROMOVERT technology with methodtriplex-forming nucleotides (TFOs), by a chromo-plasmid, can bind themutated DNA compound to the cell. In fact, knocking in genes poses a greaterchallenge than knocking them out. Like qRT-PCR, Chromovert relies on usingfluorogenic Molecular Beacons for real-time detection of nucleic acidsequences and may be applied to detect any gene of interest. Unlike qRT-PCR,Chromovert operates inside the context of living cells. Fluorescence-activatedcell sorting (FACS) to isolate positive cells. Chromovert uses MolecularBeacons and cell sorting to detect and isolate cells expressing one or moregenes. Chromovert enabled the production of a cell line for the previously outof-reach abg-ENaC ion channel.

کلیدواژه ها:

Gene therapy ، CRISPER-Cas۹ ، Chromovert ، multi-gene cell line ، flow cytometry