Transfection precipitation

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. 

Although the mechanism remains undetermined, the injection of naked DNA into skeletal muscle has demonstrated relatively high transfection efficiency. In this setting, DNA is precipitated onto the surface of microscopic metal beads (e.g., gold) and the microprojectiles are accelerated and penetrate intact tissue to several cell layers. 

Tumor tissue has also been demonstrated to take up naked pDNA following direct intratumoral injection, but this ability may be dependent on tumor type and the pDNA construct. In an important study by Vile and Hart (1993), mice bearing subcutaneous (s.c.) B16F1 melanoma or Colo 26 colon carcinoma were injected intratumorally with naked P -gal pDNA or P -gal pDNA/calcium phosphate precipitates. The tumors were collected on days 2, 4, 6 and 10 after the pDNA intratumoral injection. A gradual increase in blue-staining cells was found in the transfected melanomas with 10-15% of the cells expressing /3-gal by day ten. In contrast, none of the colon carcinoma tumors was positive for /3-gal. One explanation for the lack of in vivo transfection of the colon carcinoma is that the /3-gal pDNA constmcts contained melanoma-specific promoters (tyrosinase and TRP promoters). This study demonstrated that using an appropriate promoter established tumors could take up and express naked pDNA. 

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. 

The best known transfection technique using chemicals is the calcium phosphate method. In this method, calcium chloride and sodium phosphate are mixed together with DNA. Calcium phosphate crystals are formed upon combination of the chemicals and these crystals bind to and precipitate the DNA onto the cells in a... 

This method is extremely sensitive to pH changes which can lead to inconsistent transfection efficiencies, especially when using homebrew transfection buffers. To some extent, this sensitivity can be limited by the use of commercially available kits containing chemicals and buffers that have undergone quality control procedures, ensuring better reproducibility of results and less lot-to-lot variation. Although the costs per transfection for this method are unrivaled, the attractiveness of calcium phosphate precipitation has declined over the past 15 years, partly due to the trickiness of the method itself, the limited transfection efficiencies, and the narrow cell spectrum for which it is suitable, and partly because more modem and efficient DNA delivery methods have emerged. 

DEAE-dextran. Like the calcium phosphate co-precipitation method, the DEAE-dextran technique was originally developed to increase the viral infectivity of animal cells, and its application was later extended to transfection processes. Although it is simple, efficient, and appropriate for transient expression, its use for stable transfections has not given satisfactory results. The transfection efficiency of this method can be increased by treating cells with glycerol or DMSO. The DNA is incorporated by endocytosis, and thus exposed to extreme pH levels and cellular nucleases, which may explain, to a certain extent, the high frequency of mutations observed when transfecting by this method (Calos et al., 1983). This transfection technique can be applied to both adherent and suspension cell lines. For detailed transfection protocols, the works by Keown et al. (1990) and Kaufman (1997, 2000) are recommended. 

Jordan M, Schallhorn A, Wurm FM (1996), Transfecting mammalian cells optimization of critical parameters affecting calcium phosphate precipitate formation, Nucleic Acids Res. 24 596-601. 

Method B required for some cells which are difficult to transfect. Remove the medium from the flask and add 1 ml of the DNA precipitate. Incubate 37°C for 45 min and then add 4 ml growth medium. 

Cochleates can also be used for plasmid delivery. A negatively charged phospholipid such as phosphatidylserine, phosphatidic acid or phosphatidyl glycerol, in the absence or presence of cholesterol, are utilized to produce a suspension of multilamellar vesicles containing plasmids, which are then converted to small unilamellar vesicles by sonication. These vesicles are dialyzed against buffered divalent cations (e.g. calcium chloride) to produce an insoluble precipitate referred to as cochleates. Cochleates have been shown to encapsulate plasmid and enhance plasmid stability and transfection efficiency. 

Calcium phosphate co-precipitation, first developed by Graham and van der Eb (9), is popularly used for both stable and transient transfections in many different cell lines due to easy availability of component materials and their low cost. DNA when mixed with calcium chloride and added in a controlled manner to a buffered saline-phosphate solution at room temperature (RT) results in the formation of a precipitate that is dispersed on the attached cells (10). The precipitate is presumably taken up by the cells by endocytosis. Calcium phosphate, like several other nucleic acid delivery reagents, protects the nucleic acid in the complex from the action of intracellular and serum nucleases. 

Prepare ten 15-ml conical tubes each with 2.5 ml 2x HBS to transfect the 20 plates (25ml 2x HBS total). Transfect two plates at a time by adding 2.5 ml of DNA mixture to a tube of 2.5ml 2x HBS, mix, aud iucubate for Imiu at room temperature to allow precipitate to form. Add 5 ml of media to the tube aud mix. (Total volume iu the tube is 10ml.) Pipette 5ml outo each of the two plates. Repeat for the remaiuder of the plates. 

Add the DNA to 52ml of 2x HBS, mix well, and incubate for 1 min at room temperature to allow precipitate to form. Add the transfection mix to 1,000 ml of prewarmed DMEM-Complete Media. Discard media from cell factory. Pour media with transfection mix into the cell factory. Repeat for all cell factories. 

Other methods for quantifying the amount of DNA delivered to cells often require the use of DNA isolation procedures. Radiolabeled plasmid can be isolated from transfected cells using a modified Hirt lysis procedure (120). This procedure selectively precipitates host genomic DNA using high-salt and cold SDS precipitation, leaving plasmid in the supernatant for further purification and analysis. 

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