READ ABOUT TRANSFECTION
Tecrea feels that transfectioneers, such as yourself, should know the background literature on transfection. Transfection is a complex process involving the transfer of large molecules across biological barriers. Below is a partial list of articles which we feel maybe helpful in your reading about transfections. These articles are not about HappyFect, but they are nevertheless important as they help to appreciate the relative strengths and weakness of different transfection technologies. We hope you find the list helpful. Please join the group and contribute articles to the list.
Transfection is a group in Biological Sciences on Mendeley.
Akhtar, S. & Benter, I., 2007. Toxicogenomics of non-viral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and specificity. Advanced drug delivery reviews, 59(2-3), pp.164-82. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17481774 [Accessed March 10, 2012].
Abstract: RNA interference (RNAi) is an evolutionary conserved cellular process for the regulation of gene expression. In mammalian cells, RNAi is induced via short (21-23 nt) duplexes of RNA, termed small interfering RNA (siRNA), that can elicit highly sequence-specific gene silencing. However, synthetic siRNA duplexes are polyanionic macromolecules that do not readily enter cells and typically require the use of a delivery vector for effective gene silencing in vitro and in vivo. Choice of delivery system is usually made on its ability to enhance cellular uptake of siRNA. However, recent gene expression profiling (toxicogenomics) studies have shown that separate from their effects on cellular uptake, delivery systems can also elicit wide ranging gene changes in target cells that may impact on the “off-target” effects of siRNA. Furthermore, if delivery systems also alter the expression of genes targeted for silencing, then siRNA activity may be compromised or enhanced depending on whether the target gene is up-regulated or down-regulated respectively. Citing recent examples from the literature, this article therefore reviews the toxicogenomics of non-viral delivery systems and highlights the importance of understanding the genomic signature of siRNA delivery reagents in terms of their impact on gene silencing activity and specificity. Such information will be essential in the selection of optimally acting siRNA-delivery system combinations for the many applications of RNA interference.
Akinc, A. et al., 2005. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. The journal of gene medicine, 7(5), pp.657-63. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15543529 [Accessed March 6, 2012].
Abstract: The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm.
Chaturvedi, K. et al., 2011. Cyclodextrin-based siRNA delivery nanocarriers: a state-of-the-art review. Expert opinion on drug delivery, 8(11), pp.1455-68. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21867463 [Accessed April 6, 2012].
Abstract: INTRODUCTION: The discovery of synthetic small interfering RNA (siRNA) has led to a surge of interest in harnessing RNA interference (RNAi) technology for biomedical applications and drug development. Even though siRNA can be a powerful therapeutic drug, its delivery remains a major challenge, due to the difficulty in its cellular uptake. Naked siRNA has a biological half-life of less than an hour in human plasma. To increase the lifetime and improve its therapeutic efficacy, non-viral vectors have been developed. As a natural evolution, cyclodextrins (CDs), which are natural cyclic oligosaccharides, have recently been applied as delivery vehicles for siRNA, and this in turn, has led to a surge of interest in this area. AREAS COVERED: This review discusses the recent advances made in the design of delivery strategies for siRNA, focusing on CD-based delivery vectors, because these have demonstrated clinical success. The methods of preparation of CD-based vectors, their characterization, transfection efficiencies, cellular toxicity, preclinical and clinical trials are also addressed, as well as future therapeutic applications. EXPERT OPINION: siRNA-mediated RNAi therapeutics is beginning to transform healthcare, particularly, for the treatment of solid tumors. For example, CALAA01, a targeted, self-assembling nanoparticle system based on CD complexed with siRNA has been effective in phase I clinical trials. Although siRNA therapeutics suffers from problems related to off-target effects and non-specific gene silencing, these problems can be overcome by reducing the nanoparticle size, improving the targeting efficiency and by modifying the primary sequence of the siRNA.
Chen, C.A. & Okayama, H., 1988. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. BioTechniques, 6(7), pp.632-8. Available at: http://ukpmc.ac.uk/abstract/MED/3273409 [Accessed April 6, 2012].
Chu, G., Hayakawa, H. & Berg, P., 1987. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Research, 15(3), pp.1311-1326. Available at: http://nar.oxfordjournals.org/cgi/content/abstract/15/3/1311 [Accessed April 6, 2012].
Abstract: A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable marker.
Erbacher, P. et al., 1998. Chitosan-Based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability, Springer Netherlands. Available at: http://www.springerlink.com/content/x6225kt23r3k71p0/ [Accessed April 6, 2012].
Abstract: Purpose . Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed.
Felgner, P.L., 1987. Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure. Proceedings of the National Academy of Sciences, 84(21), pp.7413-7417. Available at: http://www.pnas.org/cgi/content/abstract/84/21/7413 [Accessed April 6, 2012].
Abstract: A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.
Fischer, D. et al., 1999. A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity, Springer Netherlands. Available at: http://www.springerlink.com/content/y0rycdr8w59qfvck/ [Accessed April 6, 2012].
Abstract: Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency.
Gao, X. & Huang, L., 1991. A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochemical and Biophysical Research Communications, 179(1), pp.280-285. Available at: http://dx.doi.org/10.1016/0006-291X(91)91366-K [Accessed April 6, 2012].
Abstract: A novel cationic derivative of cholesterol, 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), has been synthesized and used to prepare sonicated liposomes with dioleoylphosphatidylethanolamine. This novel cationic liposome reagent facilitates efficient DNA mediated transfection in A431 human epidermoid carcinoma cells, A549 human lung carcinoma cells, L929 mouse fibroblast cells, and YPT minipig primary endothelial cells. The activity was greater than that of a commercial reagent, Lipofectin, and was approximately 4-fold less toxic than Lipofectin when assayed with A431 cells. The reagent is easy to synthesize and stable for at least 6 weeks.
Gilmore, I.R. et al., 2004. The design and exogenous delivery of siRNA for post-transcriptional gene silencing. Journal of drug targeting, 12(6), pp.315-40. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15545082 [Accessed April 6, 2012].
Abstract: RNA interference (RNAi) is a natural cellular process that effects post-transcriptional gene silencing in eukaryotic systems. Small interfering RNA (siRNA) molecules are the key intermediaries in this process which when exogenously administered can inhibit or “silence” the expression of any given target gene. Thus, siRNA molecules hold great promise as biological tools and as potential therapeutic agents for targeted inhibition of disease-causing genes. However, key challenges to the effective and widespread use of these polyanionic, macromolecular duplexes of RNA are their appropriate design and efficient delivery to cells in vitro and in vivo. This review highlights the current strategies used in the design of effective siRNA molecules and also summarises the main strategies being considered for the exogenous delivery of siRNA for both in vitro and in vivo applications.
Haensler, J. & Szoka, Francis C., 1993. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chemistry, 4(5), pp.372-379. Available at: http://dx.doi.org/10.1021/bc00023a012 [Accessed April 6, 2012].
Hollins, A.J. et al., 2007. Toxicogenomics of drug delivery systems: Exploiting delivery system-induced changes in target gene expression to enhance siRNA activity. Journal of drug targeting, 15(1), pp.83-8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17365277 [Accessed March 15, 2012].
Abstract: Synthetic siRNAs are typically formulated with drug delivery systems (DDS) that improve cellular uptake for optimal gene silencing activity. Here, we show that two PAMAM dendrimer DDS, differing only in their structural architecture, elicit many different gene expression changes in human cells including opposing effects on the expression of epidermal growth factor receptor (EGFR), a gene targeted for silencing by siRNA. Despite providing similar improvements in siRNA uptake, these two formulations led to a approximately 10-fold variation in anti-EGFR siRNA activity. These data show that gene expression changes induced by DDS, separate from their ability to enhance cell uptake, determine “apparent” siRNA potency and thus offer the possibility of tailoring delivery system-siRNA combinations for additive or synergistic effects on gene silencing.
Kawai, S. & Nishizawa, M., 1984. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol. Cell. Biol., 4(6), pp.1172-1174. Available at: http://mcb.asm.org/cgi/content/abstract/4/6/1172 [Accessed April 6, 2012].
Abstract: A new procedure for DNA transfection has been developed in a system of chicken embryo fibroblast cells and cloned Rous sarcoma virus DNA by using a polycation reagent as a mediator to adsorb DNA to the cell surface and dimethyl sulfoxide as an agent to facilitate the uptake of adsorbed DNA by the cells. In this new, simple, and convenient polycation-dimethyl sulfoxide transfection, which requires no carrier DNA even with small amounts of DNA, the number of transformed cell foci induced by Rous sarcoma virus DNA was proportional to the dose of the transfecting DNA, and chicken embryo fibroblast cells were successfully transformed by v-src-containing subgenomic DNA as well.
Liu, F., Song, Y. & Liu, D., 1999. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene therapy, 6(7), pp.1258-66. Available at: http://ukpmc.ac.uk/abstract/MED/10455434 [Accessed April 6, 2012].
Abstract: Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.
Luo, D. & Saltzman, W.M., 2000. Enhancement of transfection by physical concentration of DNA at the cell surface. Nature biotechnology, 18(8), pp.893-5. Available at: http://dx.doi.org/10.1038/78523 [Accessed April 6, 2012].
Abstract: Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA-vector (i.e. DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.
Rose, J.K., Buonocore, L. & Whitt, M.A., 1991. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. BioTechniques, 10(4), pp.520-5. Available at: http://ukpmc.ac.uk/abstract/MED/1867862/reload=0 [Accessed April 6, 2012].
Abstract: One of the most efficient systems for the expression of genes in the cytoplasm of animal cells utilizes a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase. Cells infected with this virus are transfected with plasmid DNAs containing the gene to be expressed under T7 promoter control. The major limitation of this system is the efficiency with which DNA is introduced into the cell. Recently, a cationic liposome-mediated transfection reagent has yielded transfection frequencies of greater than 80%. To determine if commercially available cationic lipids could form liposomes that would yield similar transfection efficiencies, we tested liposomes prepared with five different cationic lipids. When used at appropriate concentrations in liposomes that also contained a neutral lipid, four of the five cationic lipids were effective in the transfection of HeLa cells. However, liposomes formed with the neutral lipid and one of the cationic lipids, dimethyldioctadecylammonium bromide (DDAB), gave transfection frequencies of greater than 95% and had a broad spectrum of effectiveness on a variety of cell lines. Liposomes containing DDAB are an inexpensive, highly efficient and reproducible alternative for the transfection of animal cells and are well suited for use with the vaccinia virus/T7 expression system.
Taniyama, Y. et al., 2002. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene therapy, 9(6), pp.372-80. Available at: http://ukpmc.ac.uk/abstract/MED/11960313 [Accessed April 6, 2012].
Abstract: Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripher…
Xu, Y. & Szoka, F C, 1996. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry, 35(18), pp.5616-23. Available at: http://dx.doi.org/10.1021/bi9602019 [Accessed April 6, 2012].
Abstract: To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.
