Calcium phosphate transfection

The calcium phosphate method was first used in 1973 to introduce adenovirus DNA into mammalian cells [3]. DNA-Calcium-phosphate complexes are formed by mixing DNA in a phosphate buffer with calcium chloride. These complexes adhere to the cell membrane and enter the cytoplasm by endocytosis. Disadvantages of DEAE-dextran and calcium phosphate transfection are a certain level of cytotoxicity, a complicated transfection procedure, and the fact that not all cell types can be transfected using these methods. 

Cells are transfected using the calcium phosphate transfection system as follows Prepare 1 mL of calcium phosphate—DNA suspension per 10 cm dish of cells. In a polypropylene tube, add 0.5 mL IX HBS and 10 pL phosphate solution. In a second polypropylene tube, add 20 pg of plasmid DNA (in water)... 

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. 

Batard, P., Jordan, M., Chatellard, P. and Wurm, F.M. (2001) Transfer ofhigh copy number plasmid into mammalian cells by calcium phosphate transfection. Gene 270 61-68. 

This review describes methods to experimentaUy measure chemokine receptor signaUng pathways. Standard techniques such as ceU culture han-dhng, choice of ceUs, and transfection methods are not considered here. Please note that the very cheap calcium phosphate transfection method used for transfection of COS-7 cells has been described previously (Jensen RosenkUde, 2009). Furthermore, if working with transiently transfected ceUs using the calcium phosphate transfection method, the assays are carried out 2 days posttransfection, whereas commercial miceUe-based transfection... 

Calcium-phosphate transfection has been used transiently to introduce plasmids containing the luciferase gene into animal cells (15) and electroporation has proved useful for plant and Dictyostelium cells, (16,26). For monkey CV-1 cells,10 yg of DNA was optimal for high levels of expression and the activity dropped sharply when DNA below or above this amount was used. In contrast, for electroporation of carrot cells (32) the signal increased almost linearly up to the highest amount of DNA used (50 yg/ml). It would be advisable to determine this optimal amount of DNA for other cell lines or transfection protocols. Extracts for luciferase activity are prepared and assayed as described (15,16). The Instruments used to measure activity in extracts must be calibrated periodically with purified luciferase of known specific activity to obtain an idea of the absolute amount of the enzyme being produced. For experiments in which luciferase and CAT genes are cotransfected into cells, the extracts should be made in the luciferase extraction buffer because CAT is stable in this buffer, whereas luciferase exhibits lower activity in the CAT extraction buffer. Furthermore, in such cotransfection experiments, the relative levels of CAT and luciferase expression have been found to depend upon the amounts of the two plasmids used and there is also the possibility of competition between the promoters on the two plasmids. Hence it is advisable to optimize the absolute amounts as well as the ratios of the two plasmids in such experiments. 

Composite NPs such as calcium phosphate NPs have also gained interest for nmi-viral gene delivery. Cao et al. [93] used calcium phosphate (CP) nanocomposite particles to encapsulate DNA for their delivery into MSCs. The CP-DNA NPs ( 100 nm) were reported to be less cytotoxic and more efficient in gene delivery into MSCs than those of Lipofectamine or a standard calcium phosphate transfection kit. 

S. P. Wilson, F. Liu, R. E. Wilson and P. R. Housley Optimization of calcium phosphate transfection for bovine chromaffin cells relationship to calcium phosphate precipitate formation. Anal. Biochem., 226, 212-220 (1995). 

Though several supplier offer commercial kits, many users prefer to prepare their own buffers for calcium phosphate transfections. This adds some time for preparation, sterile filtration and testing. However, if suitably stored, the solutions ate stable for years, so large quantities can be prepared at one time. A disadvantage of the method is that the crystal formation is extremely sensitive to differences in pH, which directly aflfects the solubility. Nevertheless, the method has been used successfully by many groups and has even shown potential for application at large scale (> 10 L). ... 

Jordan M. Transient gene expression in mammalian cells based on the calcium phosphate transfection method. In Al-Rubeai M, ed. Cell Engineering. Vol. 2. Kluwer Academic Publications,... 

Notes With calcium phosphate transfection methods, typically 1-5% of cells will take up and express the transfected DNA. Thus, the remaining untransfected cells serve as negative controls. 

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. 

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

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. 

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. 

Expression levels of the mutant thrombin receptors were determined in transiently transfected COS cells by assaying the level of surface expressed thrombin receptors by FACS analysis, usually for mutant receptors that showed a significant diminution of function. COS cells were transfected by calcium phosphate-DNA co-precipitation30 with 10 ng of DNA. The transfected cells were analyzed by FACS 48 h after transfection. A monoclonal antibody specific for the amino terminal region was used as the primary antibody to detect thrombin receptors present on the surface of the cells.31 A FITC conjugated goat anti-mouse IgG was used as a secondary antibody. Expression levels of the mutant receptors were determined relative to expression levels of wild-type controls. Expression-level data (% of control) for some of the mutant receptors are listed in Table 2 (wild-type thrombin... 

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. 

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