Transfection electroporation

Unmodified PNAs have been introduced to cells by microinjection [33], electroporation [60, 62, 76], co-transfection with partially hybridized DNA oligos [43, 61,... 

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

As an alternative to the chemical transfection systems, the primary cells can be effectively transfected by electroporation (Appendix 5). Amaxa has developed a nucleofection (electroporation) system that is very effective for delivering expression vectors into cells at high efficiency and low cellular mortality. The step-by-step conditions for the transfection of numerous primary cell types, including human airway epithelial cells, can be found at http //www.amaxa.com/primary-cells.html. 

The DNA or cDNA library is then introduced into a preparation of bacterial host cells. Usually, the first host selected is a laboratory strain of E. coli which has been grown and pretreated with inorganic salts to make uptake of DNA easier. The ability to take up foreign DNA is called competence, cells which have been specially prepared for the purpose are called competent cells. Other methods to transfer DNA into cells include electroporation (application of an external electric field to permeabUize the cell wall), transfection (where a recombinant bacterial virus is used to transfer the DNA to the target cell) or ballistic methods (by using DNA-coated particle projectiles). The last method has been used to introduce foreign DNA into plant cells and mammalian cells. 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

The floxed stop cassette of the integrated construct can be deleted in ES cells by transient transfection with a Cre-expressing plasmid (available, e.g., at http //www.addgene. org/) and subsequent low-density plating as selection for recombined clones is impossible. After electroporation, we plate 500-2,000 cells on feeders in a 10 cm dish and analyze 24 clones for recombination by PCR or Southern blot. At least two recombined clones should be analyzed for knockdown efficiency. 

The simplest nonviral gene transfer system in use for gene therapy is the injection of naked plasmid DNA (pDNA) into local tissues or the systemic circulation (88, 100). Naked DNA systems are composed of a bacterial plasmid that contains the cDNA of a reporter or therapeutic gene under the transcriptional control of various regulatory elements (101, 102). In recent years, work in several laboratories has shown that naked plasmid DNA (pDNA) can be delivered efficiently to cells in vivo either via electroporation, or by intravascular delivery, and has great prospects for basic research and gene therapy (101). Efficient transfection levels have also been obtained on direct application of naked DNA to the liver (103, 104), solid tumours (105), the epidermis (106), and hair follicles (106). 

In a similar study by Yang and Huang (1996), melanoma tumors in mice were in vivo transfected by the direct intratumoral administration of naked pDNA, resulting in reporter gene expression for up to ten days. In another study, Walker 256 carcinoma tumors in rats were found to express CAT after intratumoral injection of naked pDNA encoding CAT (Nomura et al., 1997). More recently electroporation has been used to deliver naked pDNA to tumors (described further in the following section). 

In one of the first studies of in vivo delivery of pDNA using electroporation, Titomirov etal. (1991) demonstrated that the skin of newborn mice could take up and express a reporter gene after pDNA injection followed by the application of electric pulses. In a later study, Heller et al. (1996) injected the liver of rats with pDNA encoding / -gal followed by the administration of electric pulses. In the rat study, long-term expression of / -gal occurred after pDNA electroporation (up to day 21 post pDNA injection) and 30-35% of the hepatocytes were transfected. 

The mechanism by which electroporation enhances i.m. pDNA expression and efficacy requires further study. For muscle transfection, it has been demonstrated that electroporation appears to increase the number of muscle fibers transfected by as much as ten-fold (Dupuis et al., 2000). In addition, the number of muscle nuclei containing transfected pDNA is higher after electroporation. The mechanism by which electroporation enhances transfection of tumor tissue is unclear at this time. 

Transfection, DNA uptake in eukaryotic systems, often is more problematic then bacterial transformation the mode of DNA uptake is poorly understood and efficiency is much lower. In yeast, cell walls can be digested with degradative enzymes to yield fragile protoplasts, which are then able to take up DNA. Cell walls are resynthesized after removal of the degrading enzymes. Mammalian cells take up DNA after precipitation onto their surface with calcium phosphate [Fugene 6 (Roche) Lipofectin (Life Technologies) Effectene (Qiagen)]. Electroporation is often more efficient for transfection in eukaryotic cell systems, especially in yeasts. 

Techniques such as electroporation and transfection have successfully been used for hybridoma production, but are much less commonly used than cell fusion assisted by polyethylene glycol. 

Transfection efficacy of naked DNA can be increased by physical methods such as electroporation and sonication. Electroporation employs electric pulses to punch holes in the cell membrane, usually smaller than 10 nm but larger than oligonucleotides. With the use of electroporation, DNA was delivered into the cytosol of cells by diffusion. Since its introduction in 1982, in vivo transfection has been achieved in skeletal muscle, fiver, skin, tumors, testis, and the kidney. Tsujie et al. (2001) developed a method to target glomeruli using electroporation in vivo wherein injection of plasmid DNA via the renal artery was followed by application of electric fields. The kidney was electroporated by sandwiching the organ... 

In another report, electroporation of ND-1 cells for transfection of EGFP gene was conducted via an aperture (2-6 pm) fabricated on a Si-SU-8-glass chip. Electroporation was performed by applying an electric pulse (10 V, 100 ms) [905], A review on microchip cell electroporation has been published [906]. 

Spencer SC (1991), Electroporation technique of DNA transfection, In Murray EJ (Ed.), Methods in Molecular Biology, vol. 7, Humana Press, Clifton, pp. 45-52. 

Teshigawara K, Katsura Y (1992), A simple and efficient mammalian gene expression system using an EBV-based vector transfected by electroporation in G2/M phase, Nucleic Acids Res. 20 2607-2611. 

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