A flow cytometry technique for measuring chromosome-mediated gene transfer

Using artificial chromosome expression systems (ACes), we have developed a unique and rapid screening technique to quantify delivery of foreign DNA into cells in vitro. Delivery was measured within 24 h after transfection, using flow cytometry to detect the transfer of ACes labeled with thymidine analogue. This technique can be used to optimize delivery parameters of ACes and heterologous DNA into cells and eventually tissue.

Chinese hamster ovary (CHO) cells carrying artificial chromosomes were grown in media supplemented with iododeoxyuridine (IdUrd). The 60-mb artificial chromosome was purified by flow cytometry sorting and transfected into Chinese hamster lung fibroblast cells (V79-4) or mouse connective tissue cells [LM(tk-)] using LipofectAMINE 2000trade mark, a cationic lipid, and Superfecttrade mark, a cationic dendrimer. The cells were incubated with an FITC-conjugated anti-bromodeoxyuridine (BrdUrd) antibody and analyzed by flow cytometry. IdUrd-incorporated artificial chromosome expressing green fluorescent protein (GFP) was transfected into V79-4 cells. Delivery was measured at 24 h and GFP expression was detected at 48 h.

The delivery of intact artificial chromosomes into V79-4 and LMtk- cells was detected within 2 h and up to 48 h post-transfection. Maximum delivery rates of 20% and 14% were observed using LipofectAMINE 2000 and Superfect, respectively. Flow cytometry data correlated with microscopic observations. IdUrd incorporation resulted in less quenching after staining with Hoechst 33258 and chromomycin A3 than BrdUrd incorporation. The fluorescence intensity of the FITC-conjugated anti-BrdUrd antibody was greater with IdUrd-incorporated chromosomes than with BrdUrd-incorporated chromosomes.

The results indicate that IdUrd-labeled artificial chromosomes can be detected 24 h after transfection. This efficient, sensitive, high-throughput detection technique is being used to evaluate and optimize other transfer technologies (e.g., electroporation and sonoporation), different delivery reagents, and protocols in a variety of cells in vitro. This work represents the first step in utilizing artificial chromosomes as nonviral vectors for gene therapy.