Calcium phosphate technique

This is a procedure increasingly used to introduce DNA into cells and various methods are available. I have experience with the calcium phosphate technique which is described below but other techniques are described by Gorman (1985) and Spandidos and Wilkie (1984). For example, Bethesda Research Ltd. supply liposomes which will mediate the uptake of nucleic acids at high efficiency (Feigner and Ringold, 1989) and with practice and the appropriate apparatus material may be injected directly into cells (Ansorge and Pepperkok, 1988). Electroporation is particularly useful for introducing DNA into plant spheroplasts. 

Chicken embryo fibroblasts (CEFs) are obtained from embryonic day-10 chicken embryos, and cultured as described (Albertinazzi et al, 1998). Transient expression of proteins is achieved by transfection of CEFs by the calcium phosphate technique using 10 fig of plasmid DNA for each 6 cm diameter plate. 18-24 h after transfection, cells are extracted with lysis buffer (1% Triton X-100,150 mM NaCl, 0.5 mg/ml phenyl methyl sulfonyl fluoride (PMSF), 20 mM Tris-Cl, pH 7.5). Cell lysates are clarified by differential centrifugation for 10 min at 18,000g in a refrigerated microcentrifuge, and utilized for analysis by sucrose velocity gradient centrifugation. 

The calcium phosphate technique is probably at present the most widely used transfection method. For calcium phosphate-mediated transfection the DNA is coprecipitated with calcium and phosphate ions. The DNA-binding capacity of the precipitate is high and under optimized conditions (crystal diameter 1 pm) the particles can contain up to 30% of their mass in the form of DNA. For best resirlts, calcium should be used in excess (125 mM), while... 

Human osteoblast-like MG63 cells were cultured on the macroporous chitosan scaffolds reinforced with hydroxyapatite or calcium phosphate invert glass were fabricated using a thermally induced phase separation technique. 

Modem production of elemental phosphoras uses a technique similar to the metallurgical processes described in Chapter 20. Apatite is mixed with silica and coke and then heated strongly in the absence of oxygen. Under these conditions, coke reduces phosphate to elemental phosphoms, the silica forms liquid calcium silicate, and the fluoride ions in apatite dissolve In the liquid calcium silicate. The reactions are not fully understood, but the stoichiometry for the calcium phosphate part of apatite is as follows ... 

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. 

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. 

Other coating processes involving fluoridated apatite have been investigated to improve the long-term adhesion and promote osteointegration of cementless titanium-based metal implants pulsed laser deposition, electron beam deposition and ion beam sputter deposition techniques, and sol-gel methods, for example. They lead to fluor-containing calcium phosphates (apatites in most cases) with different compositions and crystallinity states. 

H. Zheng, K.K. Chittur, W.R. Lacefield, Dissolution/reprecipitation of calcium phosphate thin films produced by ion beam sputter deposition technique. Biomaterials 20(1999) 443- 51. 

As the pH of milk is reduced, the colloidal calcium phosphate (CCP) dissolves and is completely soluble at pH 4.9 (Chapter 5). pH adjustment, followed by dialysis against bulk milk, is a convenient and widely used technique for varying the CCP content of milk. As the concentration of CCP is reduced, the properties of the micelles are altered but they retain some of their structure even after removing 70% of the CCP. Removal of more than 70% of the CCP results in disintegration of the micelles into smaller particles (aggregates). 

Titrations of artificially prepared mixtures containing phosphate, calcium, citrate, and sometimes proteins have been employed to study the precipitation of calcium phosphate and the inhibitory effect of citrate thereon (Boulet and Rose 1954 Eilers et al 1947 Wiley 1935). The technique is valuable for basic studies because the composition of the system can be controlled. 

Physical methods of gene transfer. Genes can often be transferred without the use of a cloning vehicle. This is especially important for certain plant cells, such as those of cereal grains, for which transfer of genes via the Ti plasmid has been difficult.167 If DNA, which may be in a plasmid, is coprecipitated with calcium phosphate, it can often be taken up directly either by animal cells or by plant protoplasts.168 169 Polycations also facilitate DNA uptake cationic liposomes seem to be especially effective.170 In the widely used electroporation technique a short electrical pulse of a few hundred volts / cm is applied to create transient pores in the plasma membrane through which the DNA can enter a cell.111 8,171 175 Chromosomes can be transferred by cell fusion and either... 

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... 

Differential 31P 1H CPMAS technique has been applied to study mineral crystals of calcium phosphate deposited during osteoblast calcification in cell culture.198 The samples harvested after 8 days of culture represent the minerals formed at the very early stage of calcification, in... 

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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