Feb 24, 2015 chemicals linear polyethylenimine lpei mw 25. For a summary of transfection efficiency results with pei at different concentrations and compared to other commercially available transfection agent, please see the results section below. Aug 22, 2019 the pdna and pei concentrations at the transfection time point were adjusted to 24 g pdna and 45 g pei per ml according to the preliminary experiments of the doe study. In this report, we describe the application of pei in the transfection of postmitotic rat sympathetic neurons in serum free, gliafree conditions, thereby showing the capability of pei as a method for studying the effects of targeted genetic manipulation in a rigorously defined cell culture system. Transfection guide overview of transfection methods promega. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. This study presents optimized protocols for lentiviral vector production using pei mediated transfection involving media that lack serum, resulting in significant cost savings. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
The invention concerns a method of synthesising and preparing linear polyethylenimine pei for use as a transfection vector, and the product obtained with such a method. Why or how does serum reduce the transfection efficiency. Serum free hek transfection media is formulated to support transfection of hek293 cells for recombinant protein and antibody expression in suspension culture under serum free conditions. Transfection of insect cell lines using polyethylenimine. Evaluation of these parameters can be done more conveniently using transfection.
In a 15cm poly propylene tube prepare the following transfection solution. Highlevel and highthroughput recombinant protein production. High cell density transient transfection of cho cells for tgf. Transfection mediated toxicity usually leads to changes in gene expression, even in the absence of any visual toxicity. Pei is the basis of most commercially available transfection agents and alone acts as a very cost effective transfection vector. Polyethylenimine pei, a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. Enhancing polyethylenimines delivery of plasmid dna into. The 2 gram bottle of pei powder can be used to make a few liters of transfection reagent, so depending on which protocol is used the cost can be about 0. Serumfree hek transfection media is formulated to support transfection of hek293 cells for recombinant protein and antibody expression in suspension culture under serumfree conditions. Universal transfection reagent is used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardtotransfect primary cells. Hi, i tried to transfect my hek293t cells with pegfp using lipofectamine 3000, following manufacturers instructions. Pegliposome complexes are stable for at least 16 hours in serum.
This protocol describes the steps needed for successful transfection of hek293 cells adapted to serumsupplemented or serumfree medium in suspension culture. Some companies provide optimized serum free transfection media to be used in combination with the. Mar 25, 2014 at the day of transfection, mix the lentiviral plasmids in 1 ml of pbs. Statistical analysis was carried out by graphpad version 5. Polyethylenimine pei transfection protocol virushost. Transfection medium is a serumfree and animalfree formulation that facilitates vector dna uptake by the insect host cells. Each system contains sufficient reagents for 40 highefficiency transfections of cells plated in 100mm tissue culture plates.
Transfection medium is a serum free and animalfree formulation that facilitates vector dna uptake by the insect host cells. Transfection protocols vary based on cell type to be transfected, transfection method, and transfection reagent. Calcium phosphate transfection is the method of choice to produce longterm stable transfectants. Transfection protocols often require serumfree conditions for optimal performance. When doing transfection, does the medium need to exclude serum. For a summary of transfection efficiency results with pei at. Transfection of mammalian cells using linear polyethylenimine. Preparation of pei transfection agent and dna binding analysis. Plate cells to obtain 7080% cell density on the day of transfection. May i know how presence of serum affects the efficiency.
Add pei to the diluted dna and vortex for 10 seconds immediately. Serumfree, animalfree formulation works seamlessly with esf 921 and esf af. However, i could only reach at most 2030% transfection efficiency. Serum resistant and enhanced transfection of plasmid dna by. It is a linear watersoluble polymer, and is attracted to negatively charged phosphate groups on dna with which it forms stable aggregates. Make sure your plasmid dna is clean, sterile, and free from phenol and sodium chloride. Peiprohq is a highly qualified version of peipro dna transfection reagent. Imagej software was used to create a statistic of the size of the complexes. Polyethylenimine pei or polyaziridine is a polymer with repeating unit composed of the amine group and two carbon aliphatic ch 2 ch 2 spacer. Transient mammalian cell transfection with polyethylenimine pei. Directly before transfection optionally the medium may be exchanged by fresh medium with or without serum containing 50 m deferrioxamine.
Linear pei was equally effective in transfection of xdc293 cells and hela cells in media containing either fetal calf serum or calf serum data not shown. The transfected gene expression can be monitored 2448 hours transient expression or 1015 days stable expression after the transfection. Change medium 24 hour later and then harvest the virus 7296 hours later. Serumfree optimem was used to directly produce lvvs that could be harvested. With a number of transfection reagents, the efficiency is reduced in the presence of fetal serum. Cells should be seeded a day prior to transfection in 6 well plates at a density of 300,000 cells per well. Method for manufacturing linear polyethylenimine pei for. Factors influencing transfection efficiency thermo fisher. Serumfree media that support cell growth, transfection and protein expression for pei mediated transient transfection can better meet regulatory compliance and simplify downstream product purification. Mem was used to directly produce lvvs that could be harvested. Furthermore, pei mediated transfections have been reported to be more consistent than capmediated transfections pham et al. Up to 90% in many cell types, including difficulttotransfect cells, primary, and stem cells easily transfect from 2 x 10 4 cells to 6 x 10 6 cells per reaction. Harvest virus from media and cells at 120 hours post transfection a.
However, studies testing serum versus serum free media have shown that higher titers could be obtained in serum containing media durocher et al. Luciferase expression was assayed 48 h after transfection using a conventional luciferase assay. Serum free media that support cell growth, transfection and protein expression for pei mediated transient transfection can better meet regulatory compliance and simplify downstream product purification. Transfection of hek293t cells with pei bowdish lab.
Transfection parameters for suspension culture can be very different to ones in adherent cells. Pei transfection of 293 cells caltech clover center. Optimizing medium components for polyethylenimine mediated. Jetpei, a transfection reagent from polyplus, is classified as a cationic polymer, with a positive charge on approximately 1 in 3 nitrogen species on its molecule. From a healthy 80% confluent 9cm plate, split in the morning 1. This study presents optimized protocols for lentiviral vector production using peimediated transfection involving media that lack serum, resulting in significant cost savings. Realize higher titers by using a formulation designed to work with esf 921 and esf af. Peg in vivo transfection protocol pegylated liposome description. The transfected gene expression can be monitored 2448 hours transient expression or. Furthermore, peimediated transfections have been reported to be more consistent than capmediated transfections pham et al. Enhanced gene silencing through human serum albumin. Linear pei is also effective in serum free media s. Suspension hek293 cells were seeded at 1 x 10 6 cellsml in serum free medium and transfected with peipro, pei max and lpei 25 kda polysciences, warrington, pa resuspended at 1 mgml. The common causes of transfection failure are decrease transfection efficiency and decreased cell viability.
Linear polyethyleneimines contain all secondary amines, in contrast to branched peis which contain primary, secondary and tertiary amino groups. Simplified lentivirus vector production in proteinfree media. Jetpei transfection reagent from polyplus transfection. A novel reaction chamber provides a dramatic increase in transfection efficiency and cell viability. Peipro, dna transfection reagent for virus production.
Appropriate for pei or lipofection mediated transfection. These properties are consistent with the higher serum protein resistance and buffer capacity of pegylated pei histidine at endosomal acidic ph. Using the ptt vector, 20 mgl of purified histagged seap was recovered from a 3. Polyethylenimine pei has been used widely in transient gene expression studies of mammalian cells. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspensiongrowing cells was more efficient with linear pei and was not affected by the presence of medium conditioned for 24 h. To improve transfection of plasmid dna as well as serum protein stability of polyionic complex nanoparticles of branched polyethyleneimine bpei, polyeth. Most transfection protocols have been developed and optimizedfor adherent culture. Transfection activity was thoroughly investigated in traditional 2d. Some companies provide optimized serumfree transfection media to be used in combination with the. Transfection with linear 25 kda reagent for 6 well dish. We performed transient gene expression in suspension chinese hamster ovary cells using a onestep transfection procedure in which dna and pei were simultaneously added to a cell culture in suspension without prior peidna complex incubation. We have defined the optimal conditions for pei based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore cl channel activity in a cf mouse model.
Our universal transfection reagent is a unique formulation of a proprietary polymer blend. Use the table below to troubleshoot transfection experimental failure. Fast and easy protocol is compatible with serum, serum free medium and antibiotics. L of proteinfree basal medium containing no growth factors, serum, antibiotics or other proteins. Among them, polyethylene imine pei is considered the gold. Cationic lipids and polymers also form complexes with dna through electrostatic interactions. Branched polyethylenimine mw 25 kda and human serum albumin were. Pegylated cationic lipid liposomebased in vivo transfection reagent is a lipidfunctionalized peg complex proprietary formulation optimized for in vivo delivery of mirna, sirna, plasmid dna and proteins. This method also works well for transient expression of. How cationic lipid mediated transfection works specially designed cationic lipids, such as the invitrogen lipofectamine transfection reagents, facilitate dna and sirna delivery into cells chesnoy and huang, 2000. Enhanced gene transfection efficiency by lowdose 25 kda. Add 3 ul of pei 1mgml stock in water per ug of dna and mix by vortexing briefly. Transfectionmediated toxicity usually leads to changes in gene expression, even in the absence of any visual toxicity.
Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral. For example, the most commonly used pei, catalog nr. Wash cells 3 x with pbs, change medium to complete cnt medium without serum transfection efficiency for mouse keratinocytes is 25 % preparation of pei stock solution dissolve pei powder to a concentration of 1 mgml in water which has been heated to 80c. Cotransfection protocol for plasmid dna and sirnamirna. Pei ratio to transfect 293f cell to generate vlp cull culture. Despite using less dna, the peimediated transfection method. Iron iii citrate inhibits polyethyleniminemediated. This protocol describes the steps needed for successful transfection of hek293 cells adapted to serum supplemented or serum free medium in suspension culture using linear pei. Optimization of lentiviral vector production using polyethylenimine. Peidna formation affects transient gene expression in. Using green fluorescent protein gfp as reporter, fcs was eliminated from the transfection process by altering critical parameters and by substituting serum with albumin. Pei is the basis of most commercially available transfection agents and acts as a very cost effective transfection vector. A comparison of linear and branched polyethylenimine pei. Two main parameters can be optimized and investigated in a transfection procedurethe level of heterologous protein expression and the transfection ef.
Here, the different cell densities, as indicated in figure 3, were transfected by the same amounts of pdna and pei table 1, and the cultures were diluted to 1. Peipro largescale virus production polyplustransfection. Factors influencing transfection efficiency thermo fisher scientific. Successful transfection is influenced by many factors such as call health and. Cells were transfected with alexafluor 555 sirnalabeled. The media has been tested for its ability to support transfection of highdensity suspension cultures of hek293ebna cells adapted from adherent culture platforms. Upon dilution in serumfree medium, the size of any kind of polyplex. Transfection of hek293ebna1 cells in suspension with linear. How cationic lipid mediated transfection works thermo. Pei is the basis of most commercially available transfection agents and alone. Calcium phosphate transfection optimization for serumfree. What i found as my answer, serum starvation synchronizes cells in g0g1 stage of the cell cycle.
Furthermore, dodecylation of primary amino groups of 2kda pei yields a nontoxic polycation whose transfection efficiency in the presence of serum is 400 times. Refeed with 2% cubate for 2h prior to transfection. Fast and easy protocol is compatible with serum, serumfree medium and antibiotics. Dmem basic medium, optimem medium, calf serum, trypsin and.
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