504 “Chimeric sorting” as an effective tool for selection of genetically modified dermal fibroblasts to be used in fundamental or applied skin research

Abstract

A technology has been developed for efficient isolation of a stable pool of HIF1a overexpressing mouse fibroblasts using a high-throughput selection of cell clones on a cellulose carrier. The pHIF1a-CBD-PDGFR genetic construct has been generated containing a EYFP reporter, a target sequence of HIF1a gene (a key factor of adaptation to hypoxia in wound healing and regeneration) and a CBD/PDGFR chimeric gene construct. The presence of CBD (cellulose-binding domain) / PDGFR (platelet-derived growth factor receptor) chimeric construct in protein-producing cells enables their specific binding to cellulose carrier thus providing a possibility for cell selection based on their adhesive phenotype (“chimeric sorting”). For genetic modification, a line of immortalized postnatal mouse dermal fibroblasts was used (dp3T3cell line). This cell line has been developed using 3T3 protocol and combines the basic features of primary dermal fibroblasts with the experimental advantages of immortal cells. The high-throughput selection of HIF1a overexpressing cells was performed using BD FACSAria™ Fusion Cell Sorter (with EYFP as a reporter) or by chimeric sorting on day 5th after dp3T3 cell transfection (electroporation) using half of transfected cells for each approach (0.9±0.2x10ˆ6). The CBD-PDGFR driven chimeric sorting was performed on cellulose filter papers, 75g/cm2. In 3 weeks chimeric sorting allowed to obtain 3±1x10ˆ6 stably transfected cells with changed morphology (less stellate and more elongate), while FACS sorting yielded 8 colonies with 0.6±0.3x10ˆ5 cells each with only 3 colonies exhibiting modified phenotype. Thus, “chimeric sorting” of transfected fibroblasts on a cellulose carrier appeared to be more effective in selection of HIF1a overexpressing cells as compared to conventional FACS. The advantageous efficiency of this approach was reflected in both its specificity (low level of contamination with cells of original phenotype) and its productivity (higher yield of stably transfected cells).