Cloned genes can be transfected into cells for biochemical characterization. Apr 12th 2012 bowdish lab, mcmaster university hamilton, on, canada. Novel polyethyleniminederived nanoparticles for in vivo. Pei transfection of 293 cells california institute of. Transfection guide overview of transfection methods. This protocol is appropriate for two suspension cell lines, chos and hek 293 gnti. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
Polyethylenimine pei or polyaziridine is a polymer with repeating unit composed of the amine group and two carbon aliphatic ch 2 ch 2 spacer. In search for a cheap and effective transfection reagent we used the positively charged polyplex. Hela cell viability after 72hours with br pei 800d, dextran and pll. You would usually preform experiments, 15 days after transfection. Assuming that the size and shape of the peidna complex is one of the most determinative variables for the high transfection ef. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Hela transfection information and resources hela cell line. Hela cells transfected by organelle light, 3d volume rendering. Recent data, however, suggest that linear polymers of the. The peidna ratio that produced the highest luciferase activity upon transfection into hela cells was found to be around 5.
Mammalian cell transfection is a technique commonly used to express exogenous dna or rna in a host cell line for example, for generating rnai probes. Transfection of dna into eukaryotic cells is a common method to study biological mechanisms. Plasmid linearization changes shape and efficiency of transfection. I have used both fugene and jetprime as transfection reagents in hela cells. Is polyethylenimine pei transfection reagent suitable in the hela. I want to transfect hela cells with lipofectamine plus reagent to optimise the quantity of dna and. The internalization route resulting in successful gene expression. A major goal is the efficient and specific delivery of genes into the desired target cells.
Pei max 40k also known as pei 22k in free base is a powerful, trusted, and costeffective transient transfection reagent. Impact of polyethylenimine conjugation mode on the cell. Pei is the basis of most commercially available transfection agents and alone. Is polyethylenimine pei transfection reagent suitable in. The effects of transfection reagent polyethyleneimine pei. Transfection efficiency of pei polyplexes in the presence of endocytosis inhibitors. Transfecting primary and stem cells basic mechanism of transfection evaluating transfection performance striking the expressiontoxicity balance optimizing transfection output making. We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine pei. Transfection is the process by which nucleic acids are introduced into eukaryotic cells. Invitro cytotoxicity of polyethyleneimine on hela and vero cells. We use these cells inhouse for testing our production. A simple and rapid nonviral approach to efficiently. Even though hela cells are very hardy, the hela cell line is fairly easy to transfect and very receptive to viral vectors, making it a popular research tool in virology.
Primary cell transfection while immortalized cell lines traditionally have served as the common targets of transfection, primary cells are often preferred as they more closely resemble the invivo nature of. Many cell lines can be transfected successfully with pei but in our. On day 8, peitreated cells had the highest viable cell density with. Pei transfection fiona whelan and charles yin last updated. Here we have optimized the peimediated transfection. Polyethylenimine pei transfection of mammalian cells in culture. Transient mammalian cell transfection with polyethylenimine pei. Thus, transfection techniques and protocols serve as an. This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available offtheshelf cationic polymer, 25kda branched. Carefully transfer the transfection mix to the lentix 293t packaging cells. Here, we describe methods for both small and largescale transient expression in mammalian cells using polyethylenimine pei. In addition, when both hela and xdc293 cells were by our method, hela cells in the absence of e1a generated threefold more recombinant aav than xdc293 cells, which constitutively express e1a.
Alternatively, the dnapei transfection complexes were performed in fresh medium 110 culture volume and then added to the cells indirect method. Efficient transfection method for primary cells tissue. I performed polyethyleniminebased transfection in hek293 cell line with an. To obtain the highest transfection efficiency, optimize transfection conditions by varying hela cell density and amount of transfection reagent. Linear polyethyleneimines contain all secondary amines, in. Add dnapei mixture to cells, after removing the medium 7. Pei transfections with gfp were equivalent or better than fugene in all 4 cells types, in particular hela cells. Transient plasmid dna transfection into hela and hacat cells. Our laboratory uses pei over other cell transfection reagents because of its low cost. Pei is the basis of most commercially available transfection agents and acts as a very cost effective transfection vector. Most methods working effectively for cell lines in. An important technical note is that, similar to a previous report optimizing pei for the transfection of hela cells, reconstituted pei should be kept frozen until ready for usage, as the efficiency of transfection. Transfection of hek 293 ebna cells could be inhibited or completely diminished if such critical components were present at other steps of the process.
Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. We find this procedure to be more costeffective and quicker than the more. A simple and efficient method for transfecting mouse. High passage of hela cells and use of antibiotics or. Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Impact on biological research and informed consent duration. Add the diluted pei dropwise while gently flicking the diluted dna tube.
Im planning to do some transient transfection of hela cells. Electroporation protocol for hela cells transfection protocol protocol no. Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Hi, our viromer red is a peiderived reagent and works perfectly fine with hela and a549cells 6570% of efficiency for plasmid transfection, 80% for mrna. Although a wide panel of techniques and vectors viral and nonviral have been developed that work with variable efficiency, most vectors lack a target cell specificity.
As a result, the transfection procedure is identical to the. Peipro largescale virus production polyplustransfection. A, b, g, h huh7, c, d cos7, and e, f, i, j hela cells were pretreated. In hek293 and cho expression systems, pei offers consistently high gene expression on a wide scale 96 well plates up to 100 l bioreactors. For the transfections with linear pei, circular and linearized plasmid dna 0. Inflammation and apoptosis related genes are upregulatedactivated due to pei transfection. Change the culture medium 2 hours after transfection. Transient plasmid dna transfection into hela and hacat. Linear pei is a better transfection reagent than calcium phosphate for the production of recombinant aav in both hek293 and hela cells.
Using various chemical or physical methods, this gene transfer technology enables the study of gene. Transient transfection in mammalian cells springerlink. Transfection of mammalian cells using linear polyethylenimine is a. Fluorination on polyethylenimine allows efficient 2d and 3d cell. Our linear pei protocol is as effective as commercial reagents in the transfection of hela cells and xdc293 cells, a derivative of hek293 cells. Additional protocols can be found in this issue, including transfection of adherent hek293ebna1 cells in a sixwell plate with branched pei for production of recombinant proteins and transfection of. Gutamic acid nanoparticles for high efficient sirna and.
Our linear pei protocol is as effective as commercial reagents in the transfection of hela cells and. In addition to transfect cells with plasmid dna carrying a specific gene. Mirus bio reagents enable high efficiency transfection of stem cells and other hard to transfect cell types used for stem cell research. I have the same problem when i used pei to transfect the hela and a549 cells, the. The critical raw materials needed for cgmp vector production must be sourced from approved suppliers and should have gone through a rigorous testing program to reduce the risk of introducing adventitious agents into the production process. Techniques vary widely and include lipidbased transfection and physical methods such as electroporation. Polyethyleneiminemediated transfection of cultured. Our linear pei protocol is as effective as commercial reagents in the transfection of hela cells and xdc293 cells, a derivative of hek293 cells, but at a fraction of the cost. We found that the most successful approach was to add the dnatransfection mix to 80% confluent hela cells for 5 hours then gently remove the media. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Principles and mechanisms of mammalian cell transfection. Evaluation of transfection methods for transient gene.
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