May 29, 2008 transfection with polyethylenimine pei was evaluated as a method for the generation of recombinant chinese hamster ovary cho dg44 cell lines by direct comparison with calcium phosphate. This technique involves mixing your dna construct with calcium chloride in a buffered saline phosphate solution. Calcium phosphate transfection of packaging cells quantities for one 15cm plate. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum. Transfection with polyethylenimine pei was evaluated as a method for the generation of recombinant chinese hamster ovary cho dg44 cell lines by direct comparison with calcium phosphate. In animal cells, transfection is the preferred term. The critical factor in capo4 coprecipitation transfections is the ph of the solutions. Calcium phosphate transfection is a commonly used method for the introduction of dna into eukaryotic cells.
Calcium phosphate an overview sciencedirect topics. Chemical transfection transfections using chemical transfection reagents rely on electrostatic interactions to bind with nucleic acids and to target cell membranes. The protocol is versatile and, although used primarily on adherent cells in culture, also can be modified to transfect cells growing in suspension 10. Surface functionalization of cap nanoparticles cap nps with n2aminoethyl3aminopropyltrimethoxysilane was shown recently to achieve dispersed nps with a positive surface charge, capable of transfection chem. Twentyfour hours prior to transfection, inoculate 1 to 2 x106 cells10 cm plate or, inoculate 3x106 cellsplate 16hours prior to transfection. Dna coprecipitation capo4 using both green fluorescent protein gfp and a monoclonal antibody as reporter proteins. Drosophila schneider 2 s2 cells thermo fisher scientific. Calcium phosphate transfection kingston 2003 current. Mechanisms underlying this transfection method are not yet defined. However, calcium phosphate coprecipitation is prone to variability and is not suited for in vivo gene transfer into whole animals.
Calcium phosphate transfection kit protocol sigmaaldrich. Calcium phosphate transfection protocol utilize the following mix components for all experiments. Upon oral uptake, calcium phosphate competes for and blocks the absorption of radium ra226 and strontium sr90 in the gastrointestinal gi tract. This technique has been used to obtain both transient and stable transfections in a wide variety of cell types. Amounts refer to a transfection of one p100 of cells 1 precipitate 1020 g of dna. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Calcium phosphate biomaterials are well known in the medical materials and devices world, in particular, for their use in the treatment of osseous defects and as coatings on metal alloy.
Calcium phosphate cap is the main mineral found in human bone and teeth and is considered as a highly biocompatible inorganic biomaterial legeros, 2008. Calcium phosphate transfection is one of the most commonly used methods to deliver plasmid dna into mammalian cells. Calcium phosphate transfection is one of the chemical transfection methods using this principle. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. Drosophila schneider 2 s2 cells can be transfected with the recombinant expression vector alone for transient expression studies or in combination with a selection vector e. Whether such microprecipitates are formed and whether they are relevant to the transfection remain open questions. Calcium phosphate transfection of mammalian cultured cells. Calcium phosphate magnetic nanoparticles have been successfully developed by puddu et al. This unit contains two methods of calcium phosphate based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. Calcium phosphate transfection operates effectively in hek cells, which is the most commonly used cell culture transfection model. High efficiency production of lentiviral vectors in hek 293ft.
At the time of transfection, cells should be about 3040%. We developed quantitative flow cytometry to analyze the correlations between dna uptake and other parameters known to influence transfection efficiency exposure time to plasmidcapi. Calcium phosphate transient transfection protocol and guide. Many methods have been developed in order to use calcium phosphate cap for delivering nucleotides into living cells. In these protocols, plasmid dna is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. The calcium phosphate transfection method for introducing dna into mammalian cells is based on forming. Early transfection studies used viral rna and dna 2, 4, which, at that time, were relatively easy to propagate and purify, and allowed.
Calcium phosphate transfection current protocols wiley. Pdf calcium phosphate transfection generates mammalian. Transfer of high copy number plasmid into mammalian cells. The calcium phosphate transfection protocol is routinely used for both transient and stable transfection of a variety of cell types. Calcium phosphate and deaedextran reagents were incorporated into the first chemical methods that successfully transferred nucleic acid directly to cultured mammalian cells in a process referred to as transfection 1, 2, 3, 4. This can be achieved with compounds like calcium phosphate, polycations, and liposomes or more current technologies such as cationic lipids, polymers, dendrimers, and nanoparticles. This study optimized the protocol of calcium phosphate transfection for mscs and highlighted the importance of serum during the process of transfection. At the time of transfection, cells should be about 3040% confluent. This protocol is a modified version of a published method4, in which calcium phosphate based transfection methods for chinese hamster ovary cells and the 293 line of. The following protocol is written for transfection of cells in a 10cm. Briefly, lentivirus was packaged by co transfection of larlbgfp construct with the packaging and envelope plasmids pspax2 and pmd2. The uptake and fate of dna transfected into mammalian cells in vitro.
Transfect as soon as they are adherent and 7585% confluent. The procedure is based on slow mixing of hepesbuffered saline containing sodium phosphate with a cacl 2 solution containing the dna. In this report, we optimized the protocol of calcium phosphate transfection for murine mscs, and compared this protocol with other gene transfer methods in different. Dna calcium phosphate coprecipitates arise spontaneously in supersaturated solutions. Transient assays for gene expression in transfected cells are performed 4872hrs post transfection. Transfection can be carried out using calcium phosphate i. The procedure is based on slow mixing of hepesbuffered saline containing sodium phosphate with a cacl 2 solution. Calcium phosphate facilitates the binding of the dna to the cell surface. The ph should be checked with nonbleeding sterile ph sticks or reliable ph meters. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below. Lenti virus can be packaged by any transfection reagent. Supplied reagents are sufficient to perform 1 control and 75 transfection reactions.
Calcium phosphate transfection has been optimized and widely used with. Calcium divalent ions bind the dna polymers and neutralize their negatively charged phosphate backbones. Transfection at 70% confluency is suitable for both transfection methods. Transfectopedia methods transit certified transfection. High efficiency production of lentiviral vectors in hek. For those experiments where more transfection mix is needed, simply use a multiple of the reagents. Transfection guide overview of transfection methods.
The positively charged lipids associate with the negatively charged phosphate groups of genetic materials. Calcium phosphatemediated dna transfection springerlink. Transfection system of aminofunctionalized calcium phosphate. Calcium phosphate transfection is the method of choice to produce longterm stable transfectants.
Calcium phosphatetransfection kit thermo fisher scientific. Product components and storage conditions product cat. For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with l7rhbeta. This unit contains two methods of calcium phosphate. Calcium phosphate transfection works very well for 293 cells up to 95% transfection efficiencies. With optimization, it is possible to use this method on hardtotransfect cells like primary neurons. Surface functionalization of cap nanoparticles cap nps with n2aminoethyl3aminopropyltrimethoxysilane was shown recently to. For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with l7rhbetagal plasmid. Calcium phosphate transfection thermo fisher scientific. Application calcium phosphate transfection kit has been used.
Calcium phosphatemediated transfection of eukaryotic. Transfer of high copy number plasmid into mammalian cells by. Transfection can result in unexpected morphologies and abnormalities in target cells. Sep 17, 2019 calcium phosphate transfection is an inexpensive and welldescribed method without discernible biosafety issues. The imfectin method requires a small amount of plasmid dnas and is simpleefficient. C and 5% co 2 and split 110 every few days so they do not become over confluent. Mixing dna with calcium chloride in a phosphate buffer. Calcium phosphate transfection of 293t cells 2 transfection 1. Calcium phosphate transfection kingston 1999 current. High efficiency production of lentiviral vectors in hek 293ft cells using plasmid co transfection. Calcium phosphate transfection optimization for serumfree. Calcium phosphate is the calcium salt of phosphoric acid with widely used applications. For those experiments where more transfection mix is. This method also works well for transient expression of transfected genes and can be used with most adherent cell lines.
This step generates a precipitate that is dispersed onto the cultured cells. In our study on calcium phosphate transfection, we used both ethidium monoazide and pna labelling and we analyzed plasmid uptake in individual cells. Utilize the following mix components for all experiments. This method relies on the use of positively charged calcium ions to bind to the negatively charged phosphate backbone of dna and form a coprecipitation complex that can then be added to. However, rapid particle growth has largely prevented the translation of this method for in vivo purposes. Calcium divalent ions bind the dna polymers and neutralize their negatively charged phosphate. Difference between transfection and transformation. Factors that can be optimized for calcium phosphate transfections are presented in. Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. Some cells are calcium tolerant and can live in the presence of ca overnight, while other cells are calcium intolerant and should be exposed to the precipitate for no longer than 46 hours. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physicochemical conditions that control the initiation and growth of precipitate complexes. Currently, there is no optimized protocol for mscs.
Calcium phosphate transfection thaler center for hiv and. Detailed instructions for the transfer of the rphyco erythrin protein into. Calcium phosphate transfection of eukaryotic cells. Calcium phosphate transfection kit most cost effective. Depending on the time of assay, plate sparsely or more densely. A reduction of the calcium concentration from 250 mm to 100 mm, yielded a 25fold increase in the expression of the recombinant protein compared to the serumfree standard conditions. Aug 05, 2016 calcium phosphate based transfection is a standard and well known technique. Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection. Principle dna is mixed with calcium chloride addition to buffered salinephosphate solution and incubating at room temperature formation of dnacalcium phosphate coprecipitates which adhere to surface of cells. The calcium phosphate transfection is an inexpensive and simple. The critical factor in capo 4 coprecipitation transfections is the ph of the solutions.
Calcium phosphate transfection of primary hippocampal neurons. Both methods of transfection require very highquality plasmid. Calcium phosphate transfection of primary hippocampal. Calcium phosphate based transfection is a standard and well known technique. The uptake and stability of simianvirus40 dna after calcium phosphate transfection of cv1 cells.
Aug 05, 2016 calcium phosphate transient transfection protocol. It may also refer to other methods and cell types, although other terms are often preferred. Protocol for lentivial vector lv production 2nd generation. Nonadherent cell lines can be transfected by electroporation and liposome mediated transfection. Transfection of adherent and suspended cells by calcium. This agent can be used as a countermeasure for exposure to strontium and radium radionuclides. The calcium phosphate transfection method for introducing dna into mammalian cells is based on forming a calcium phosphate dna precipitate. The most common transfection methods are chemicalmediated transfection such as calcium phosphate coprecipitation, liposomes, and physical methods such as electroporation, gene gun technique, and microinjection.
This technique has been used to obtain both transient 1 and stable 2 transfections in a wide variety of cell types. In the protocols, plasmid dna is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. All solutions in the fivephoton biochemicals calcium phosphate transfection kits are provided sterile and adjusted to ph 7. Since the solutions are of low ionic strength, often 2 or 3 ph meters must be consulted to correctly determine the ph. Calcium phosphate transfection is wellestablished and safe, but the protocols need to be optimized according to different cell types. The dna to be expressed is cloned into the pmt2t or related vector which contains a sv40 origin, adeno major late promoter, the adeno tripartite leader and the va rna i gene, leading to very high level expression of the recombinant protein. G by calcium phosphate transfection into hek293t cells. The calcium phosphate transfection is a widely used method for introducing foreign dna plasmids into cells. Principle dna is mixed with calcium chloride addition to buffered saline phosphate solution and incubating at room temperature formation of dna calcium phosphate coprecipitates which adhere to surface of cells. When the mixture is incubated at room temperature, dna calcium phosphate coprecipitates form. Lipidmediated transfection, also known as lipofection, uses lipids with similar properties to that of cell membrane. Pdf transfection system of aminofunctionalized calcium. This unit presents two methods of calcium phosphate. Due to its highly consistent ph and salt concentration, our calphos calcium phosphate transfection kit gives you the consistent transfection efficiency that you cannot always achieve with home brew versions.
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