Transfecting DNA

The process by which cells take up large molecules is called endocytosis. Some of these molecules (eg, polysaccharides, proteins, and polynucleotides), when hydrolyzed inside the cell, yield nutrients. Endocytosis provides a mechanism for regulating the content of certain membrane components, hormone receptors being a case in point. Endocytosis can be used to learn more about how cells function. DNA from one cell type can be used to transfect a different cell and alter the latter s function or phenotype. A specific gene is often employed in these experiments, and this provides a unique way to smdy and analyze the regulation of that gene. DNA transfection depends upon endocytosis endocy-... 

As pDNA and mRNA transfection differ in both the timing of mRNA expression and the gross amount of mRNA delivered to the cell, it is important to identify a suitable time point to measure miR-mediated repression. We observe that at any time point after transfection, pDNA transfections have higher measurable levels of miR-mediated repression compared to mRNA transfections (Fig. 6.2C). This difference may, in part, reflect a time lag of active miR-protein-complex formation relative to the onset of translation of the transfected Renilla luciferase mRNA. For single time point experiments, we decided to measure miR-mediated repression in mRNA and DNA transfections at 16 and 24 h, respectively. 

Figure 6.2 Critical parameters of the miR/mRNA co-transfection method. (A) Titration of mRNA amount. HeLa cells were transfected with increasing amounts of cap tail R-luc-4 sites mRNA and a fixed amount of firefly (F-luc) mRNA. R-luc expression (luciferase activity) was measured 5 h after transfection. (B) Titration of miCXCR4 concentration. HeLa cells were transfected with cap tail R-luc-4 sites mRNA, F-luc mRNA, and varying concentrations of miCXCR4. Luciferase activity was measured 16 h after transfection and fold-repression by the miR was calculated as in Fig. 6.1D (C) Time-course of miR-mediated repression. HeLa cells were co-transfected with cap tail R-luc-4 sites and F-luc (control) mRNAs, either with or without miCXCR4, and harvested at different time points. Repression was calculated as detailed in Fig. 6.1 and plotted against time (mRNA transfection data series depicted by the circles). Analogous plasmid DNA transfections are shown for reference (pDNA, diamonds). Averaged results from several experiments are shown with standard deviation. Data were previously published (Humphreys etal., 2005). Copyright PNAS, reprinted with permission.

Pinnaduwage, P., Schmitt, L., and Huang, L., Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells, Biochimica et Biophysica Acta, 1989, 985, 33-37. 

Zauner W, Kichler A, Schmidt W, Sinski A, Wagner E (1996) Glycerol enhancement of ligand-polylysine/DNA transfection. Biotechniques 20 905-913... 

Malek A, Czubayko F, Aigner A (2008) PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy. J Drug Target 16 124-139... 

Sequential hydroformylation/reductive amination of dendritic perallylated polyglycerols with various amines in a one-pot procedure to give dendritic polyamines in high yields (73-99%). Furthermore, the use of protected amines provides reactive core-shell-type architectures after deprotection. These soluble but membrane filterable multifunctional dendritic polyamines are of high interest as reagents in synthesis or as supports in homogeneous catalysis as well as nonviral vectors for DNA-transfection (Scheme 18) [65]. 

Feigner PL, Gadek TR, Holm M, et al. Lipofection a highly efficient, lipid-mediated DNA transfection procedure. ProcNatl Acad Sci USA 1987 84 7413. 

In addition to extracellular degradation in tissues, endosomal acidification might also trigger PEG-lipid cleavage. We showed that despite the presence of the PEG, which slightly reduces lipoplex internalization into the cells, DNA transfection level almost reaches the level of the cationic lipo-plex (31). Cholesterol PEG incorporation into lipoplexes not only reduces lipoplex internalization, but also inhibits the transfection efficiency. 

Polymeric oxazolines have also been used as vehicles for controlled drug release ° ° and DNA transfection, as polymeric micelles, which serve as carriers for drug transport (e.g., paclitaxel), and as formulation additives for controlled-release of insecticides. ... 

Clinical trials have demonstrated excellent efficacy with recombinant human factor VIII concentrates available as Recombinate and Kogenate. These recombinant factor VIII products are purified from the cell culture of plasmids, not viral DNA-transfected hamster cells and therefore do not express viral sequences. The addition of human serum albumin for stabilization, constitutes the sole possible source for human viral contamination. More recently recombinant factor IX has been genetically engineered by insertion of the human factor IX gene into a Chinese hamster ovary cell line. It has been proved to be safe and effective in the treatment of patients with hemophilia B. 

Feigner, P.L., T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz, J.P. Northrop, GM. Ringold, and M. Danielsen, Lipofec-tion a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA, 1987. 84(21) 7413-17. 

Zhou, X., Huang, L. (1994). DNA transfection mediated by cationic liposomes containing lipopolysine characterization and mechanism of action. Biochim. Biophys. Acta, 1189, 195-203. 

Component of liposomes Protective coating for liposomes Protein stabilizer DNA transfection agent Surfactant in intranasal or ocular delivery Increasing susceptibility of tumor cells to cytotoxic drugs Liver gene delivery... 

DNA from both rat nasal squamous carcinomas (2) and mouse skin squamous carcinomas (4) and fibrosarcomas (4) arising in dimethylcarbamoyl chloride-treated animals failed to transform NIH 3T3 cells by DNA transfection (Garte et al., 1985). 

Coonrod, A., Li, F.Q. and Horwitz, M. (1997) On the mechanism of DNA transfection efficient gene transfer without viruses. Gene Ther., 4, 1313-1321. 

Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res., 11, 1295-1308. 

Feigner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M. etal. (1987) Lipofection A highly efficient, lipid mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA, 84, 7413-7417. 

Guenin, E., Herve, A.C., Floch, V., Loisel, S., Yaouanc, J.J., Clement, J.C. et al (2000) Cationic phosphonohpids containing quaternary phosphonium and arsonium groups for DNA transfection with good efficiency and low cellular toxicity.Angew. Chem. Int. Edit.,39,629-631. 

The viral protein R (Vpr) of HIV-1 plays a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in non-dividing cells. The C-terminal domain of Vpr (Vpr52-96), which condenses plasmid DNA, mediates DNA transfection in a variety of human and non-human cell lines (Table 16.7). The Vpr52-96 sequence... 

Kichler, A., Pages, J.C., Leborgne, C., Druillennec, S., Lenoir, C., Coulaud, D. et al. (2000) Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R. J. Virol., 74, 5424-5431. 

Glasspool-Malone, J., et al. 2002. DNA transfection of macaque and murine respiratory tissue is greatly enhanced by use of a nuclease inhibitor. J Gene Med 4 323. 

Ciftci, K., and R.J. Levy. 2001. Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts. Int J Pharm 218 (1-2) 81. 

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