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

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