On the mechanism of DNA transfection: efficient gene transfer without viruses

Abstract

From an investigation of how transfected DNA navigates abrogation of endosome-lysosome fusion or translocation from the cell surface to the nucleus, we have developed with microfilament or microtubule toxins, respectively, a transfection method for primary human fibroblasts that inhibits the nuclear accumulation of transfected DNA, but approaches the efficiency of viruses. We have visually interruption of lysosomal function with protease inhibitors tracked the subcellular routing of exogenous DNA and find promotes it. Second, in normal human fibroblasts, which that all cells in an asynchronous population are surprisingly are refractory to transfection, the exogenous DNA is rapidly competent in the nuclear uptake of DNA, but two steps excluded from the nucleus, but in HeLa cells, which are practically limit efficient transfection to a minority of cells.readily transfected, there is prolonged nuclear stability of First, regardless of the method used to traverse the cell the DNA, indicating the failure in HeLa cells of a mechanism membrane -CaPO 4 precipitation, lipofection or electroporfor the elimination of foreign DNA. These observations ation -it appears that nuclear transport of DNA requires imply strategies for optimizing gene transfer efficiency in routing through endosomes and lysosomes. Apparent virus-independent approaches to gene therapy.

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound

“…[8][9][10][11][12][13][14]20 Also, the absolute number of the reported transfection efficiencies might vary since there was a 24 h interval between incubation with DNA/sonication and analysis. If cell proliferation takes place, transfected and nontransfected cells might divide not neccessarily at the same rate.…”

“…Gene Therapy (2000) 7, 1516-1525. nonviral vectors have certain advantages: they might be easier to prepare, can transfect quiescent cells, lack proteins invoking immunogenic responses, and lack limits in the size of genes that can be delivered. 4,5,8 Nonviral methods mostly used for standard in vitro laboratory transfection, including electroporation and calcium phosphate coprecipitation, are not suitable for general in vivo application despite encouraging reports for superficial tissue using electroporation as a transfection method. 9 On the other hand, direct tissue injection of naked DNA 8,10 or injection of plasmids encapsulated in liposomes.…”

“…4,5,8 Nonviral methods mostly used for standard in vitro laboratory transfection, including electroporation and calcium phosphate coprecipitation, are not suitable for general in vivo application despite encouraging reports for superficial tissue using electroporation as a transfection method. 9 On the other hand, direct tissue injection of naked DNA 8,10 or injection of plasmids encapsulated in liposomes. 4,5,11,12 are reliable and simple techniques for gene transfection that may have therapeutic potential, eg in cancer therapy.…”

Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer

“…When nonviral vectors are introduced to the cell through liposomal transfections, they are endocytosed and carried via microtubules through the endosomal-lysosomal pathway and are found to accumulate around the nucleus before release into the cytoplasm. 28,29 With the method of transfection we primarily use in our lab, either electroporation or microinjection, it is unclear as to what pathways plasmids use to traverse the cytoplasm and enter the nucleus. Regardless of the process involved, it is clear that once in the cytoplasm, the plasmid must be quickly shuttled to the nucleus, since nucleases found in the cytoplasm can begin to degrade DNA in a matter of minutes.…”

Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres

“…As shown for cationic liposomes successful transfection of mammalian cells requires efficient transportation of exogenous DNA in the nuclei through endosomal and lysosomal machinery. 20 Large sizes of MF-2 particles (about 2 m) are hardly suitable for the cellular processing of this vehicle by standard intranuclear trafficking. None the less direct visualization of these microspheres proves that their size does not interfere with their efficient incorporation into the muscle and other cell nuclei.…”