A flow cytometry technique for measuring chromosome-mediated gene transfer

Background: Using artificial chromosome expression systems (ACes), we have developed a unique and rapid screening technique to quantify delivery of fo reign DNA into cells in vitro. Delivery was measured within 24 h after tran sfection, using flow cytometry to detect the transfer of ACes labeled with thymidine analogue. This technique can be used to optimize delivery paramet ers of ACes and heterologous DNA into cells and eventually tissue. Method: Chinese hamster ovary (CHO) cells carrying artificial chromosomes w ere grown in media supplemented with iododeoxyuridine (IdUrd). The 60-mb ar tificial chromosome was purified by flow cytometry sorting and transfected into Chinese hamster lung fibroblast cells (V79-4) or mouse connective tiss ue cells [LM(tk-)] using LipofectAMINE 2000(TM), a cationic lipid, and Supe rfect(TM), a cationic dendrimer. The cells were incubated with an FITC-conj ugated anti-bromodeoxyuridine (BrdUrd) antibody and analyzed by flow cytome try. 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. Results: 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 de livery rates of 20% and 14% were observed using LipofectAMINE 2000 and Supe rfect, respectively. Flow cytometry data correlated with microscopic observ ations. IdUrd incorporation resulted II 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 IdU rd-incorporated chromosomes than with BrdUrd-incorporated chromosomes. Conclusion: The results indicate that IdUrd-labeled artificial chromosomes can be detected 24 h after transfection. This efficient, sensitive, high-th roughput detection technique is being used to evaluate and optimize other t ransfer technologies (e.g., electroporation and sonoporation), different de livery reagents, and protocols in a variety of cells in vitro. This work re presents the first step in utilizing artificial chromosomes as nonviral vec tors fur gene therapy. Cytometry 44:100-105, 2001. (C) 2001 Wiley-Liss, Inc .