Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells.
We prepared a series of linearized DNA duplexes of various lengths in order to examine the effects of topology and the size of exogenous, plasmid-derived DNAs on transgene expression. These linearized DNA duplexes were capped at each end with a highly stable loop (5'-GCGAAGC-3') to produce a dumbbell-shaped construct that is refractory to exonuclease digestion in comparison to the analogous uncapped DNA duplexes. Intranuclear microinjection of the DNA dumbbells into simian COS-7 cells allowed the expression of the green fluorescent protein (GFP) gene on the linearized molecules, which was expressed five- to 10-fold more than that on the circular DNA of the same size. In addition, the expression by the dumbbell DNA was higher than that by the circular plasmid for at least 14 days. Interestingly, the size of the dumbbell DNA affected the transgene expression upon their microinjection into cell nuclei. The GFP expression efficiency increased with decreasing DNA size below a DNA size of 5.7 kb. The effects of topology and size on the expression of DNAs transfected with cationic lipids are similar to those of DNAs microinjected into cell nuclei. In contrast, microinjection into the cytosol showed the inverse size dependency over a range of 2.3 to 9.4 kb. Thus, transcription of a transgene in the nucleus, but not endocytosis or nuclear entry, was influenced by the exogenous DNA structure, and this was the primary determinant of transgene expression upon transfection under our conditions. These results indicate that small, linearized DNA duplexes that are end-capped with a highly stable loop (dumbbell-shaped DNA) would be very useful for nonviral gene therapy.
Kamiya
H
H
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Yamazaki
J
J
Harashima
H
H
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008845:Microinjections; D014162:Transfection / Q000379:methods*; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA