Metrics details. RNA interference RNAi is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target.
The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells HAoSMCs where we suspected that off target effects through this mechanism might be substantial.
Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. We have been exploring RNAi for the purpose of modifying the vascular response to injury especially during graft implantation, employing endothelial cells and vascular smooth muscle cells, under various conditions and technology for siRNA delivery [ 1 — 4 ].
Although these studies have displayed promise of RNAi therapy as an alternative means to treat vascular diseases, there is so far no study discussing how transfection reagent alone and in combination with non-targeting control siRNAs affect gene expression level.
Our group is interested in developing siRNA based therapies to treat vascular bypass grafts, both vein and prosthetic, to prevent vascular graft failure. Injury to the graft and the host artery during graft implantation is the most important trigger for the downstream graft failure. Vascular smooth muscle cells play a major role in the failure process and thus are often targeted for therapeutic intervention.
When determining the effectiveness of siRNA silencing of a target gene, the standard control has been a comparison with control siRNA. In our laboratory we have used a series of controls. For example, if using a transfection reagent, our controls would be: 1.
Saline alone. Transfection reagent alone. Transfection reagent plus control siRNA. We have noticed differences between these controls with regard to expression of the target gene, raising the question of the control effects on total genomic expression. For example, if the transfection reagent alters target gene expression and that is different from the transfection reagent plus the control siRNA, which is the better control?
Can we count on the control siRNA to have no effect on target gene expression? Should saline or balanced salt solution be the appropriate control as it is least likely to affect target gene transcription? In this study, experiments were designed to determine the global effects on gene transcription of saline, a transfection reagent, and the transfection reagent plus control siRNA using RNA sequencing.Pontiac 428 4 bolt main
Since our goal is to treat vascular bypass grafts with siRNA, we chose a transfection reagent PEI, as this is frequently used for in vivo applicability.
After treatment, cells were subjected to RNA isolation as described above. After preprocessing and normalization of RNA sequencing data we performed unsupervised analysis using PCA to determine relationship between different treatment groups as well as samples within each group.
Biological replicates from most of the groups clustered together indicating similar transcriptome profile. PEI Pwhich comes in two forms; linear and branched polymer, has been extensively used as a non-viral cationic carrier to deliver drugs or genes into the cells via proton sponge effects [ 5 ].
Out of differentially expressed genes, and 98 were significantly downregulated and upregulated respectively Fig. Colors indicate standardized values green represents down regulation and red represents up regulation. To understand the underlying biological mechanism of alterations induced due to PEI treatment, we performed gene-ontology GO categories and canonical pathways analysis. These pathways that are triggered by immune systems in response to PEI transfection, play a critical role in multiple diseases including cancer, immunological and neurodegenerative diseases.
To understand the biological mechanism underlying the alterations induced by control siRNA transfection, we performed functional and pathways enrichment analysis on the genes that were commonly altered by both Invitrogen and Dharmacon control siRNA. These pathways are the primary line of host defense against infection by bacterial pathogens and are rapidly recruited to sites of bacterial invasion suggesting that control siRNAs are probably recognized as foreign entities [ 6 ].Linear PEI is a cationic polymer commonly used for complexing DNA into nanoparticles for cell-transfection and gene-therapy applications.Speaker frequency test
The polymer has closely-spaced amines with weak-base protonation capacity, and a hydrophobic backbone that is kept unaggregated by intra-chain repulsion. As a result, in solution PEI exhibits multiple buffering mechanisms, and polyelectrolyte states that shift between aggregated and free forms. We studied the interplay between the aggregation and protonation behavior of 2. Our results indicate that:. At neutral pH, the PEI chains are associated and the addition of NaCl initially reduces and then increases the extent of association.
The aggregate form is uncollapsed and co-exists with the free chains. PEI buffering occurs due to continuous or discontinuous charging between stalled states. Ninhydrin assay tracks the number of unprotonated amines in PEI. Despite its simple chemical structure, linear PEI displays intricate solution dynamics, which can be harnessed for environment-sensitive biomaterials and for overcoming current challenges with DNA delivery.
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This material is based upon work supported by National Science Foundation under grant no. Sonya Smith. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. Linear polyethylenimine PEI is a widely used polymer for packing negatively charged DNA into nanometer-sized particles for cell delivery[ 1 — 3 ].
The polymer consists of amines separated by ethylene groups [ 45 ]:. In an acidic environment the PEI chain is positively charged; the charge comes from the protonation of the secondary amines along the backbone. It protects the DNA from the acidic environment of cell-uptake vesicles and ensures the DNA release into the cytoplasm[ 167 ]. PEI is a hydrophobic polymer because of its ethylene-rich backbone.
When there is insufficient backbone charge to keep the molecule extended by intra-chain charge repulsion, the polymer collapses or aggregates[ 89 ]. The conformation of a charged hydrophobic polymer strongly depends on the solution conditions see Fig 1 and Sec 1A below [ 10 ]. Linear PEI is the simplest hydrophobic, weak-base buffering polyelectrolyte. It is not known how the protonation of PEI is coupled with its hydrophobic-polyelectrolyte characteristics [ 11 ].
The polymer charge increases with decreasing pH, whereas the charge repulsion is screened by increasing the salt concentration.
The backbone extension depends on the balance between inter-chain and intra-chain charge repulsion. Aggregation occurs as intra-chain repulsion is lowered or as inter-chain repulsion is increased.
The backbone extension of a hydrophobic polyelectrolyte depends on the competition between inter- and intra-chain interactions Fig 1. Intra-chain charge repulsion i.
Solution conditions affect the balance between inter- and intra-chain repulsion in the following way: pH increases the charge of the polymer, while added salt reduces charge repulsion due to screening the electrostatic interaction [ 12 ]. Increasing PEI concentration increases inter-chain effects. At low-ionic strength the attractive hydrophobic interactions between the polymer segments are often counterbalanced by the electrostatic repulsion, so that an extended molecular conformation is observed.
We will review strategies within product characterization to de-risk the manufacturing process, including the use of routine and high complexity assays; and the recommended testing to meet regulatory requirements for clinical submission.
Learn methods to avoid costly pitfalls and implement procedures to expedite product quality decisions at critical junctures in your development plan. We will review the regulations and the liquid chromatography with charged aerosol detection LC-CAD methodology to demonstrate PEI removal during the production process.
Host cell protein HCP impurities, present at PPM-levels, are a major immunogenicity risk because they can elicit an unpredictable immune response in patients. We will review why their complex and diverse nature makes them challenging to monitor, and theho best practices, specifically HCP identification by mass spectrometry, for detection.
Learning points: 1. Accurate detection and characterization of residual PEI in cell and gene therapy products 2. Effective detection and characterization of residual host cell proteins HCP in mAbs 3.
Available technology and assays for quantifying process impurities 4. Current regulatory requirements for detecting, quantifying, and removing process impurities during biotherapy manufacturing. SlideShare Explore Search You. Submit Search. Home Explore. Successfully reported this slideshow. We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads.
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Are you sure you want to Yes No. You can also request things like research papers or dissertations. Adam Sheldon Hello! I can recommend a site that has helped me.Polyethylenimine PEI or polyaziridine is a polymer with repeating unit composed of the amine group and two carbon aliphatic CH 2 CH 2 spacer.
Linear polyethyleneimines contain all secondary amines, in contrast to branched PEIs which contain primary, secondary and tertiary amino groups. Totally branched, dendrimeric forms were also reported. Linear polyethyleneimines are soluble in hot water, at low pH, in methanolethanolor chloroform.Mammalian cell Transfection
They are insoluble in cold water, benzeneethyl etherand acetone. They can be stored at room temperature. Branched PEI can be synthesized by the ring opening polymerization of aziridine. Linear PEI is available by post-modification of other polymers like poly 2-oxazolines  or N -substituted polyaziridines. Polyethyleneimine finds many applications in products like: detergents, adhesives, water treatment agents and cosmetics.
Polyethylenimine (PEI), linear (1 mg/mL)
PEI has a number of uses in laboratory biology, especially tissue culturebut is also toxic to cells if used in excess. Polyethyleneimines are used in the cell culture of weakly anchoring cells to increase attachment. PEI is a cationic polymer; the negatively charged outer surfaces of cells are attracted to dishes coated in PEI, facilitating stronger attachments between the cells and the plate.Nexys 4 github
Poly ethylenimine was the second polymeric transfection agent discovered,  after poly-l-lysine. PEI condenses DNA into positively charged particles, which bind to anionic cell surface residues and are brought into the cell via endocytosis. Once inside the cell, protonation of the amines results in an influx of counter-ions and a lowering of the osmotic potential. Osmotic swelling results and bursts the vesicle releasing the polymer-DNA complex polyplex into the cytoplasm.
If the polyplex unpacks then the DNA is free to diffuse to the nucleus. Poly ethylenimine is also an effective permeabilizer of the outer membrane of Gram-negative bacteria. Both linear and branched polyethylenimine have been used for CO 2 capture, frequently impregnated over porous materials.
First use of PEI polymer in CO 2 capture was devoted to improve the CO 2 removal in space craft applications, impregnated over a polymeric matrix.Syringe manufacturing
The authors claim that, in this case, a synergic effect takes place due to the high PEI dispersion inside the pore structure of the material. As a result of this improvement, further works were developed to study more in depth the behaviour of these materials.
Moisture and real world conditions have also been tested when using PEI-impregnated materials to adsorb CO 2 from the air. A detailed comparison among PEI and other amino-containing molecules showed an excellence performance of PEI-containing samples with cycles. The first approach used a combination of them to impregnate porous supports, achieving faster CO 2 -adsorption kinetics and higher stability during reutilization cycles, but no higher efficiencies.Search Thermo Fisher Scientific.
Search All. A major goal is the efficient and specific delivery of genes into the desired target cells. 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. Nonviral vectors are attractive because of their ease of manipulation, safety and high flexibility in the size of the delivered transgene.
The Transferrinfection is a high-efficiency nucleic acid delivery system based on transferrin receptor-mediated endocytosis to carry DNA into cells. Furthermore it was shown that the cationic polymer polyethylenimine PEI mediates efficient gene transfer into a variety of cells.
The method results in a 30 - fold enhanced transfection efficiency depending on the cell line. It is an extremely gentle DNA transfection method whichemploys physiological uptake mechanisms of the cell. Transfection efficacy depends on the cell type, the level of surface transferrin receptor expression.
Very high transfection rates have been shown for the tested tumor cell lines B16F10 melanoma, Neuro 2A neuroblastoma and a variety of primary human melanoma cell lines. For Research Use Only. Not for use in diagnostic procedures.Metrics details. Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates.
Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines. Polyethyleneimine compared favorably to traditional attachment factors such as collagen and polylysine.
PC and HEK cells plated on dishes coated with polyethyleneimine showed a homogeneous distribution of cells in the plate, demonstrating strong cell adhesion that survived washing procedures. The polymer could also be used to enhance the adherence and allow axonal outgrowth from zebrafish retinal explants. The effects of this coating agent on the transfection of loosely attaching cell lines were studied. Pre-coating with polyethyleneimine had the effect of enhancing the transfection yield in procedures using lipofection reagents.
Polyethyleneimine is an effective attachment factor for weakly anchoring cell lines and primary cells.
Its use in lipofection protocols makes the procedures more reliable and increases the yield of expressed products with commonly used cell lines such as PC and HEK cells. Molecular cell biology experimentation often requires the culture of primary cells or immortalized cell lines. The most common substratum used in cell culture consists of a plastic dish that offers a negatively charged surface. A drawback of this technology is that some anchorage-dependent cell types do not produce sufficient amounts of positively charged extracellular matrix proteins, adhering only weakly to the plastic substratum.
Pre-coating of the plastic surface with extracellular matrix proteins such as collagen, fibronectin, laminin, etc. Synthetic polymeric cations such as polylysine or polyornithine have also been used as attachment promoting factors in numerous studies [ 34 ]. Polyethyleneimine PEI is an organic polymer that has a high density of amino groups that can be protonated. At physiological pH, the polycation is very effective in binding to DNA and can mediate the transfection of eukaryotic cells [ 5 ].
The original PEI-based protocol has been used in our laboratory successfully for transfections with HEK human embryonic kidney and PC rat pheochromocytoma cells. Ballestero, unpublished observations.Remembering your Grade 5 math teacher, you calculate that only 18. Does this mean you should immediately go bet the Blue Devils because the public clearly knows something that you don't or do you immediately bet on Wake Forest because anytime 81.
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Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer
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