In:
The Journal of Gene Medicine, Wiley, Vol. 9, No. 3 ( 2007-03), p. 197-207
Abstract:
Heterogeneity of transgene expression, the presence or absence (below the limit of detection) of transgene expression on a cell‐by‐cell basis, is a severe disadvantage in the use of cationic lipid‐mediated gene vectors for gene therapy and experiments in molecular biology. Understandings of intracellular trafficking and the function (transgene expression) of vectors related to cellular physiology are essential in terms of clarifying the mechanism underlying the heterogeneity. Methods To distinguish the contribution of nuclear transfer efficiency and subsequent intranuclear transcription efficiency to the overall heterogeneity in transgene expression, a novel imaging system was established for the dual visualization of the nuclear transfer of pDNA and marker gene expression (lacZ) in single cells. Results The expression of LacZ occurred in only approximately 30% of HeLa cells of the nuclear pDNA‐positive cells, indicating that intranuclear transcription efficiency contributed to the heterogeneity. Dual imaging against synchronized cells further revealed that the efficiency of nuclear delivery was comparable irrespective of cell cycle status, which is contrary to the generally accepted hypothesis that nuclear import of pDNA is enhanced during cell division when the nuclear membrane structure is perturbed. The most significant finding in the present study is that nuclear transcription efficiency in terms of the ratio of LacZ‐positive cells to nuclear pDNA‐positive cells drastically increased in the late S and G2/M phase. Conclusions This is the first demonstration to show that cell cycle dependent intranuclear transcription appears to be responsible for the overall heterogeneity of transgene expression. Copyright © 2007 John Wiley & Sons, Ltd.
Type of Medium:
Online Resource
ISSN:
1099-498X
,
1521-2254
Language:
English
Publisher:
Wiley
Publication Date:
2007
detail.hit.zdb_id:
2002203-7
SSG:
12
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