New strategy for rapid isolation of stable cell lines from DNA-transformed insect cells using fluorescence activated cell-sorting

Abstract

A stably transformed insect cell expression system is superior to a baculovirus expression system, since the expression system is sustained and there is no cell lysis, but the isolation of cell lines producing recombinant proteins is time-consuming and laborious. In this study, we developed a technique for the rapid isolation and efficient cultivation of sorted cells in a 24 well deep plate Bioshaker, utilizing the fluorescence activated cell-sorting (FACS) method. TnpXme11 cells, which stably expressed GFP(uv)-beta1,3-N-acetylglucosaminyltransferase2 (GGT2), were transfected with a plasmid vector for the expression of a molecular chaperone (TnpXme11-hCNX6 cell line). The expression levels of GGT2 and the molecular chaperone fused with HcRed were analyzed by FACS. Two cell lines were established by single and double sorting. Sorting of the top 10% of the TnpXme11-hCNX6 cell population with the highest fluorescence yielded the TnpXme11-hCNX6-1 cell line. TnpXme11-hCNX6-2 cells were created in a similar fashion, as mentioned above, by a second sorting of the top 10% of the TnpXme11-hCNX6-1 cell population with the highest fluorescence. The cells thus isolated produced approximately 2-fold higher extracellular activity than that before cell sorting. This procedure can be accomplished in only 2 weeks, including transfection, isolation and analysis of high protein-producing cells, and is a breakthrough strategy for the rapid isolation of a recombinant, stable insect cell line.

Comparison of Inlet Geometry in Microfluidic Cell Affinity Chromatography

“…Many techniques have been developed to realize high throughput and high-purity cell isolation and separation . These techniques can be classified into two types: separation based on internal properties, such as size, shape, and electrical properties, − or separation based on cell surface markers, such as affinity surface or matrix, fluorescence-activated cell sorting, and magnetic-activated cell sorting. − Among these methods, cell separations based upon affinity chromatography have become increasingly important in bioanalytical and diagnostic applications due to the features of rapid analysis, high selectivity, low cost, and ease of use. − …”

“…1 These techniques can be classified into two types: separation based on internal properties, such as size, shape, and electrical properties, [2][3][4][5][6] or separation based on cell surface markers, such as affinity surface or matrix, fluorescence-activated cell sorting, and magnetic-activated cell sorting. [7][8][9][10] Among these methods, cell separations based upon affinity chromatography have become increasingly important in bioanalytical and diagnostic applications due to the features of rapid analysis, high selectivity, low cost, and ease of use. [11][12][13][14] Cells can be captured by antibodies, aptamers, or other capture ligands that recognize a cell surface marker.…”

Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

“…FACS is widely used to select high-producing subpopulations of insect cells. 32,33 Instead of time-consuming cell line screening methods involving limiting dilution and colony picking, our screening method requires only sorting of the population of cells having the highest (top 10%) green fluorescence intensity using FACS (Fig. 2B).…”

Comparison of bidirectional and bicistronic inducible systems for coexpression of connexin genes and fluorescent reporters

“…Previously generated inducible systems have either lacked an in vitro reporter of expression [5] or expressed a fused reporter protein and thus expressed chimeric Cx [6][7][8]. The co-expression of a reporter gene is often essential as an indicator of vector transfection and gene expression, or as a selectable marker in lieu of antibiotic selection [9]. The terminal fusion of a fluorescent reporter can, however, modify protein structure and function; this has been shown with regard to Cx function and assembly [6,10,11].…”