Nuclear-Associated Plasmid, but Not Cell-Associated Plasmid, Is Correlated with Transgene Expression in Cultured Mammalian Cells

Abstract

Intracellular plasmid is rapidly incorporated into the nucleus of HeLa cells following cationic lipoplex transfection. CV1 cells are less effective in translocating plasmid to the nucleus and also express less transgene than HeLa cells. Cultured HeLa and CV1 cells and corresponding isolated nuclei were analyzed after transfection of a Cy3-labeled pGreenLantern plasmid (Cy3-pGL). Flow cytometry was used to measure both plasmid delivery and transgene expression from the plasmid encoding a CMV promoter-driven green fluorescent protein. During transfection, HeLa cells rapidly incorporated the plasmid, reaching a maximum of 80% Cy3-pGL positive cells 8 h posttransfection. The average Cy3-pGL-positive HeLa cell contained approximately 2470 plasmid copies. Forty-eight percent of the nuclei isolated from the transfected HeLa cells were positive for the plasmid marker after 8 h. In contrast to HeLa cells, fewer CV1 cells and CV1 nuclei incorporated plasmid DNA with peak transfection occurring after 12 h for 36% of the cells and after 8 h for 12% of the nuclei. However, the average Cy3-pGL-positive CV1 cell did not have a significantly different number of total cellular plasmid copies than the average positive HeLa cell. CV1 nuclei, however, had half as much nuclear associated plasmid as HeLa nuclei. HeLa cells are more efficient than CV1 cells at transporting plasmid from the cytoplasm to the nucleus. This study demonstrates the use of a novel quantitative method to study plasmid transport from the cytoplasm to the nucleus and the effect on transgene expression.

A model for the analysis of nonviral gene therapy

“…A mathematical representation of this compartmental model was utilized to evaluate quantitative data acquired from previous experiments performed in our laboratory. 22 These data conform to the physical and mathematical models to estimate the relative significance of specific intracellular mechanisms. Rate constants derived from our analysis of experimental data are used to characterize the mechanism/s that are responsible for heterogeneous capacity for transgene expression in two in vitro cell types, HeLa cells (human epitheliod, ATCC CCL-2) and CV1 cells (monkey fibroblast, ATCC CCL-70), and to identify possible rate-limiting steps during transgene delivery.…”

“…22 The ratelimiting barriers to nonviral gene therapy and the effects of modulated parameters such as cell type on plasmid translocation can be revealed through this quantitative approach.…”

“…22 Experimental details are described previously 22 and are not reproduced here. Techniques and nomenclature particularly germane to this work are highlighted in the Methods section that follows the Discussion.…”

“…Initial plasmid concentration added to the cells was obtained by spectrophotometry and spectrofluorescence as previously described. 22 The number of plasmids translocated from the supernate was calculated at any time as a difference between the starting plasmid number and the number of plasmids delivered intracellularly. In this way, the small reduction in extracellular plasmid can be determined with precision.…”

“…22 These measurements represent the percent of the cell population that is producing green fluorescent protein (GFP), based on the fluorescence intensity measured by flow cytometry (percent positive). At 4 h, GFP expression in terms of percent positive in HeLa cells was double that of CV1 cells.…”