Transfection methods

Viruses are infectious particles formed by nucleic acid, proteins, and in some cases lipids. As viruses (for example, retro- and adenoviruses) transfer viral genes into cells with high efficiency, modified forms are sometimes used as vectors for gene transfer. However, procedures using virus-based vectors are often significantly more complicated and time-consuming than other transfection methods. In addition, viral vectors are potentially hazardous, and biological safety issues need to be considered carefully. Therefore, techniques that combine... 

The generation number, activation procedure, and core moiety of PolyFect Transfection Reagent have been developed specifically for transfection of certain cell lines, including HeLa and COS-7. Optimized amounts of DNA and den-drimer delivered transfection efficiencies in both cell lines significantly higher than those obtained using a standard transfection method. 

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.

Transient mammalian High authenticity Relatively fast methods Scale-up difficult Transfection methods cell line-specific... 

We may also find that Sabatini s reverse transfection method of creating "live cell" microarrays offers even greater advantage for drug discovery and development (Ziauddin and Sabatini, 2001). Their method, relying on arrayed cDNA expression vectors to transform adherent cells, provides localized real-time gene expression analysis of the putative gene product. 

Expression by transient transfection methods using mammalian cell lines is a convenient and rapid method of producing recombinant proteins when E. coli systems fail to produce correctly folded, structurally homogeneous protein. Moreover, it is a method that is routinely used to produce proteins for crystallization. 

The article by Hahn and Scanlan gives a general overview of common transfection methods and presents a valuable entrance point to the field. 

Transfection methods that use a mini-Ad plasmid DNA system (Grimm et al., 1998 Grimm, 2002 Zolotukhin et al., 2002) to supply Ad helper functions and AAV genes are incapable of... 

Poston RS, Mann MJ, Hoyt EG, Ennen M, Dzau VJ, Robbins RC. Antisense oligodeoxynucleotides prevent acute cardiac allograft rejection via a novel, non-toxic, highly efficient transfection method. Transplantation 1999 68 825-832. 

The success of transfection, either stable or transient, depends on several factors that must be taken into account and can be summarized as follows (i) the transfectability and physiology of the cell line (ii) the characteristic of the genetic marker in the expression vector (iii) the type of expression desired (iv) the size of the expression cassette and the quality of the DNA to be introduced (v) the compatibility of the transfection method and/or reagents with the cell line (vi) the type of assay to be used for detection of... 

The use of linearized or circular DNA depends on the transfection method, but, in general, linearized DNA is advisable... 

In the next sections, the transfection methods most commonly used in cell culture laboratories will be described. It is difficult to predict the best method for transfection, so the expression vector, the cell type, and the facilities of each laboratory should be taken into account before deciding which technique to apply (Wurm, 2004). For infection techniques, an updated compilation can be obtained from Heiser (2004). 

The choice of transfection method varies depending on the cell type to be transfected. We generally use calcium phosphate or electroporation to transfect cells. Both methods work well on a large variety of cell types. Cationic... 

In general, transfection methods can be divided into (see Subheading 1.1.1.) physical or direct transfer methods like electroporation, high-velocity bombardment, microinjection and (see Subheading 1.1.2.) chemical methods via carriers, e.g., lipofection, calcium phosphate, and DEAE-dextran. 

Due to the vast number of DNA transfection methods and reagents available to the researcher (Table 2), it will be a hard task to describe specific protocols... 

Use a different lipofection/transfection method. This may be particularly important when signs of cytotoxicity appear. 

Befcxe the evolution of effective transfection methods and an understanding of molecular biological techniques, DNA and RNA were ad- ministered as potential therapeutic agents. For example, DNA from several sources displayed antitumor activity and the activity was reported to vary as a function of size, base i composition, and secondary structure (1-3). However, the molecular mechanisms by which DNA might induce antitumor effects were never defined and numerous other studies failed to demonstrate antitumor activities with DNA (4). 

Fourth, MIDGE vectors exhibit a feasible expression of recombinant proteins in mammalian cells. Compared to plasmids, expression rates are equal or even higher (depending on the promoter employed, the transfection method and the cell line used), probably due to their small size. A high level of transient gene expression is achieved for several hours or days, and a lower level of expression can be detected for several weeks (for up to 80 days after intramuscular injection). 

In addition to calcium phosphate transfection, methods for DNA transfer into mammalian cells by electroporation [31, 32] and by transfection mediated through cationic lipids, liposome [33-35], biohstics [36] and polymers [37, 38] have been developed. Most of these techniques have been reported to mediate higher transfection efficiencies as compared to calcium phosphate-mediated DNA transfer. Such claims must be regarded with some caution, as all DNA transfer techniques estabhshed so far suffer from high variabihty due to technical difficulties. Other factors to cause major variations in transfection efficiency are the type of cells used and the condition of the cells prior to transfection. 

Unfortunately, a reliable and comprehensive analysis of the various options for vector design has never been carried out, in part because this would be a very difficult and time-consuming task. Most comparisons of promoter/enhancer elements have been made with model proteins and in transient transfections since expression from individual clonal cell lines or from populations from stable transfections range dramatically from one experiment to another, and are also dependent on the transfection method. 

Early experiments showed that a transferrin-polycation complex transported bacterial DNA into cells [12]. Ions are taken up by cells as an iron-transferrin complex by receptor-mediated endocytosis. Protamine or poly-lysine ligated by disulfide bonds to transferring and mixed with a lu-ciferase-encoding plasmid may bind the DNA because of the cationic properties of the complex [12]. Subsequently, avian ery-throblasts and human K-562 cells were incubated with the transferrin-polycation peptide-DNA complex, and the complexes were recognized and transported into the cells by receptor-mediated endocytosis and taken up into endosome-Hke intracellular vesicles [12]. Treatment with chloroquine (an agent that affects the endosomal pH) enhanced the uptake considerably. In contrast to other transfection methods, the transfection of cells with transferrin-mediated endocytosis did not cause significant cell death, because of the physiologi-... 

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