Lipid-based Transfection Reagents Research Articles

The use of cell and larval assays to identify target genes for RNA interference-meditated control of the Australian sheep blowfly (Lucilia cuprina).

Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical-based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi-mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay. This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

STING inhibition enables efficient plasmid-based gene expression in primary vascular cells: A simple and cost-effective transfection protocol.

Plasmid transfection in cells is widely employed to express exogenous proteins, offering valuable mechanistic insight into their function(s). However, plasmid transfection efficiency in primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs) is restricted with lipid-based transfection reagents such as Lipofectamine. The STING pathway, activated by foreign DNA in the cytosol, prevents foreign gene expression and induces DNA degradation. To address this, we explored the potential of STING inhibitors on the impact of plasmid expression in primary ECs and SMCs. Primary human aortic endothelial cells (HAECs) were transfected with a bicistronic plasmid expressing cytochrome b5 reductase 4 (CYB5R4) and enhanced green fluorescent protein (EGFP) using Lipofectamine 3000. Two STING inhibitors, MRT67307 and BX795, were added during transfection and overnight post-transfection. As a result, MRT67307 significantly enhanced CYB5R4 and EGFP expression, even 24 hours after its removal. In comparison, MRT67307 pretreatment did not affect transfection, suggesting the inhibitor's effect was readily reversible. The phosphorylation of endothelial nitric oxide synthase (eNOS) at Serine 1177 (S1177) by vascular endothelial growth factor is essential for endothelial proliferation, migration, and survival. Using the same protocol, we transfected wild-type and phosphorylation-incapable mutant (S1177A) eNOS in HAECs. Both forms of eNOS localized on the plasma membrane, but only the wild-type eNOS was phosphorylated by vascular endothelial growth factor treatment, indicating normal functionality of overexpressed proteins. MRT67307 and BX795 also improved plasmid expression in human and rat aortic SMCs. In conclusion, this study presents a modification enabling efficient plasmid transfection in primary vascular ECs and SMCs, offering a favorable approach to studying protein function(s) in these cell types, with potential implications for other primary cell types that are challenging to transfect.

Surfactant like peptides for targeted gene delivery to cancer cells

Surfactant like peptides (SLPs) are a class of amphiphilic peptides widely used for drug delivery and tissue engineering. However, there are very few reports on their application for gene delivery. The current study was aimed at development of two new SLPs, named (IA)4K and (IG)4K, for selective delivery of antisense oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) to cancer cells. The peptides were synthesized by Fmoc solid phase synthesis. Their complexation with nucleic acids was studied by gel electrophoresis and DLS. The transfection efficiency of the peptides was assessed in HCT 116 colorectal cancer cells and human dermal fibroblasts (HDFs) using high content microscopy. The cytotoxicity of the peptides was assessed by standard MTT test. The interaction of the peptides with model membranes was studied using CD spectroscopy. Both SLPs delivered siRNA and ODNs to HCT 116 colorectal cancer cells with high transfection efficiency which was comparable to the commercial lipid-based transfection reagents, but with higher selectivity for HCT 116 compared to HDFs. Moreover, both peptides exhibited very low cytotoxicity even at high concentrations and long exposure time. The current study provides more insights into the structural features of SLPs required for nucleic acid complexation and delivery and can therefore serve as a guide for the rational design of new SLPs for selective gene delivery to cancer cells to minimize the adverse effects in healthy tissues.

Contaminating transfection complexes can masquerade as small extracellular vesicles and impair their delivery of RNA.

One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV‐producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid‐based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid‐based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV‐mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably‐expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.

Rationally designed cationic amphiphilic peptides for selective gene delivery to cancer cells

Gene therapy has gained increasing attention as an alternative to pharmacotherapy for treatment of various diseases. The extracellular and intracellular barriers to gene delivery necessitate the use of gene vectors which has led to the development of myriads of gene delivery systems. However, many of these gene delivery systems have pitfalls such as low biocompatibility, low loading efficiency, low transfection efficiency, lack of tissue selectivity and high production costs. Herein, we report the development of a new series of short cationic amphiphilic peptides with anticancer activity for selective delivery of small interfering RNA (siRNA) and antisense oligodeoxynucleotides (ODNs) to cancer cells. The peptides consist of alternating dyads of hydrophobic (isoleucine (I) or leucine (L)) and hydrophilic (arginine (R) or lysine (L)) amino acids. The peptides exhibited higher preference for transfection of HCT 116 colorectal cancer cells compared to human dermal fibroblasts (HDFs) and induced higher level of gene silencing in the cancer cells. The nucleic acid complexation and transfection efficiency of the peptides was a function of their secondary structure, their hydrophobicity and their C-terminal amino acid. The peptides containing L in their hydrophobic domain formed stronger complexes with siRNA and successfully delivered it to the cancer cells but were unable to release their cargo inside the cells and therefore could not induce any gene silencing. On the contrary, the peptides containing I in their hydrophobic domain were able to release their associated siRNA and induce considerable gene silencing in cancer cells. The peptides exhibited higher selectivity for colorectal cancer cells and induced less gene silencing in fibroblasts compared to the lipid-based commercial transfection reagent DharmaFECT™ 1. The results from this study can serve as a tool for rational design of new peptide-based gene vectors for high selective gene delivery to cancer cells.

WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents

Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure–activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.

DNA-binding Cell-penetrating Peptide-based TRAIL Over-expression in Adipose Tissue-derived Mesenchymal Stem Cells Inhibits Glioma U251MG Growth.

Genetic manipulation of stem cells using non-viral vectors is still limited due to low transfection efficiency. We investigated whether the DNA-binding cell-permeation peptides (CPP) can enhance the transfection efficiency of non-viral vectors in adipose tissue-derived mesenchymal stem cells (ASCs) and whether ASCs over-expressing TRAIL through CPP can inhibit the growth of glioma U251MG cells in vitro and in vivo. ASCs were genetically engineered to over-express TRAIL by using CPP, pCMV3-TRAIL and lipid-based transfection reagents (X-tremeGENE). The transfection efficiency of ASCs increased by approximately 7% using CPP; 53.9% of ASCs were transfected and TRAIL expression in ASCs increased by approximately 3 times compared to X-tremeGENE alone. ASCs over-expressing TRAIL using CPP inhibited growth of glioma U251MG cells both in vitro and in the U251MG xenograft model. CPP can be used as an enhancer for genetically manipulating ASCs and tumor treatment.

Molecular targeting of vulnerable RNA sequences in SARS CoV-2: identifying clinical feasibility

Covid-19 (SARS CoV-2) has become a deadly, world-wide pandemic. Although most who are infected survive, complications from the virus can be pronounced and long-lasting. To date, of all the respiratory viruses including influenza and coronaviruses, only influenza has had a drug (i.e., Tamiflu) specifically targeted to treat and prevent infection. As a result, additional agents that specifically target viral production and are clinically feasible are needed to alleviate respiratory viral infections. The idea of using a miRNA/siRNA molecular approach for treating various diseases was postulated over a decade ago; however, only within the past few years has it become feasible. One technological advancement has been the molecular linkage of lipophilic moieties to mi/siRNAs in order to bypass the need for enveloping these inhibitory RNAs in lipid-based transfection reagents, which could irritate the airway if inhaled. Here we show that siRNAs and miRNAs inhibit SARS CoV-2 spike protein production in a dose-dependent manner in both HEK293 cells and a primary human airway tracheal cell line. We also show that this inhibition is equally robust using a clinically relevant siRNA that does not need to be prepped with a transfection reagent.

Rational design of transient gene expression process with lipoplexes for high-level therapeutic protein production in HEK293 cells

Abstract Transient gene expression, an attractive technology that enables the rapid production of recombinant proteins for the development of biopharmaceutical candidates at an early stage, offers an interesting alternative to the time-consuming generation of stable recombinant cell lines. In this study, a highly efficient transient gene expression system was established by optimizing significant factors; namely, the host cells, lipid-based transfection reagent, enhancer, and expression vector. HEK293E and Expi293 F cells were evaluated to determine the best host cells to use, and the cell passage number and initial cell density were optimized for high transient gene expression. Cationic liposomes were selected as the vehicle for plasmid DNA delivery and optimized with respect to the co-lipid complex and DNA-to-lipid ratio. Among the cationic lipids, DC-Chol/DOPE gave a more than 2-fold increase in production yield. Following gene delivery into cells, butyric acid (a histone deacetylase inhibitor) supplementation increased protein production by a maximum of 4-folds through transcriptional regulation. Additionally, an expression vector bearing the E77 element at the 3′ end of the polyadenylation signal sequence further enhanced the human-Fc and bevacizumab yields by 7% and 15%, respectively. Finally, our novel combination method was successfully applied to human IgG and Fc protein production using Expi293 F cells.

Cholesterol depletion induced by RNA interference targeting DHCR24 protects cells from liposome-induced cytotoxicity

Existing evidence has demonstrated liposomes as the gene transporter induce the cytotoxicity during the transfection process through several known pathways. In the present study, we investigated the possibility of siRNAs targeting 3-β-hydroxysterol △-24-reductase (DHCR24), which encodes an enzyme catalyzing the last step of cholesterol biosynthesis, to suppress the liposome cytotoxicity induced by lipid-based transfection reagent in the neuroblastoma cell line N2A. We found that the siRNAs targeting DHCR24 mRNA protect cells from the liposome-induced cell death, probably through the effect of siDHCR24s on the reduction of the cellular cholesterol and decrease in the generation of reactive oxygen species (ROS). This suggests that siRNAs targeting DHCR24 or other methods that reduce the intracellular cholesterol levels might be a good strategy for avoiding the cytotoxicity of liposomes, without impairing its efficiency of gene-delivering.

Selected lipid-based transfection reagents activate NF-κB and MAP kinases signaling pathways, induced cytokines mRNA expression in human THP-1 macrophage

In the present study, we evaluated, under transient transfection conditions, five different cationic lipid-based transfection reagents on the activation of NF-κB, MAP kinases signaling pathways and induction of cytokines expression. We found that the reagents studied differentially regulated the NF-κB and the MAP kinases signaling pathways in the human THP-1 macrophage. Additionally, mRNA expression levels of the cytokines, IL-1β and TNF-α in THP-1 macrophage were also induced by selected test reagents. Hence, careful selection of cationic lipid-based transfection reagent for transient transfection is warranted, especially in studies of gene expression and function mediated through NF-κB- and MAP kinases-mediated pathways.

RNA Interference to Knock Down Gene Expression.

RNA interference (RNAi) is a biological process by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. As a tool for knocking down the expression of individual genes posttranscriptionally, RNAi has been widely used to study the cellular function of genes. In this chapter, I describe procedures for using gene-specific, synthetic, short interfering RNA (siRNA) to induce gene silencing in mammalian cells. Protocols for using lipid-based transfection reagents and electroporation techniques are provided. Potential challenges and problems associated with the siRNA technology are also discussed.

Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents.

In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identified. Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like Turbofect. Transfection efficiency could be affected by the nature of target cell line and vector. Transfection efficiency and cytotoxicity of each reagent was identified by using flow cytometry and XTT assay, respectively. Lipofectamine 3000 was more efficient in transfecting pCDH, while Turbofect was more efficient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. Lipofectamine 3000 could be cytotoxic in transfecting H9T-cells with pCDH. Also, H9T-cells were not sufficiently transfected with each plasmid vector by using each Lipofectamine 3000 and Turbofect. Turbofect had less cytotoxicity effect on all three cell lines than Lipofectamine 3000.Transfection of suspended cells like H9T-cells by using Lipofectamine 3000 and Turbofect would not result in sufficient transfection. Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 1, Fig. 2, Ref. 26).

Trans-acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high-throughput microbioreactor system.

A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS10+ as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS10+ to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS10+ -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS10+ to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS10+ -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS10+ for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

Lipid-based DNA/siRNA transfection agents disrupt neuronal bioenergetics and mitophagy.

A multitude of natural and artificial compounds have been recognized to modulate autophagy, providing direct or, through associated pathways, indirect entry points to activation and inhibition. While these pharmacological tools are extremely useful in the study of autophagy, their abundance also suggests the potential presence of unidentified autophagic modulators that may interfere with experimental designs if applied unknowingly. Here, we report unanticipated effects on autophagy and bioenergetics in neuronal progenitor cells (NPCs) incubated with the widely used lipid-based transfection reagent lipofectamine (LF), which induced mitochondria depolarization followed by disruption of electron transport. When NPCs were exposed to LF for 5 h followed by 24, 48, and 72 h in LF-free media, an immediate increase in mitochondrial ROS production and nitrotyrosine formation was observed. These events were accompanied by disrupted mitophagy (accumulation of dysfunctional and damaged mitochondria, and of LC3II and p62), in an mTOR- and AMPK-independent manner, and despite the increased mitochondrial PINK1 (PTEN-inducible kinase 1) localization. Evidence supported a role for a p53-mediated abrogation of parkin translocation and/or abrogation of membrane fusion between autophagosome and lysosomes. While most of the outcomes were LF-specific, only two were shared by OptiMEM exposure (with no serum and reduced glucose levels) albeit at lower extents. Taken together, our findings show that the use of transfection reagents requires critical evaluation with respect to consequences for overall cellular health, particularly in experiments designed to address autophagy-inducing effects and/or energy stress.

Combinational use of lipid-based reagents for efficient transfection of primary fibroblasts and hepatoblasts.

Commercially available lipid-based transfection reagents are widely used to deliver DNA to cells. However, these lipid-based transfection reagents show poor gene transfer efficiency in primary cells. Here, we demonstrate a simple method to improve gene transfer efficiency in primary fibroblasts and hepatoblasts using a combination of lipid-based transfection reagents. Our data show that combined use of Lipofectamine LTX and FuGENE HD increases the efficiency of gene transfer compared with the use of either reagent alone, and this combination achieves the best result of any pairwise combination of Lipofectamine LTX, FuGENE HD, TransFectin, and Fibroblast Transfection Reagent.

Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency.

The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

Lipid-based transfection reagents can interfere with cholesterol biosynthesis

Lipid-based transfection reagents are widely used for delivery of small interfering RNA into cells. We examined whether the commonly used commercial transfection reagents DharmaFECT-4 and Lipofectamine 2000 can interfere with lipid metabolism by studying cholesterogenesis. Cholesterol de novo synthesis from [14C]acetate was assessed in human hepatocyte-derived Huh-7 cells. The results revealed that DharmaFECT, but not Lipofectamine, markedly inhibited cholesterol biosynthesis by approximately 70%. Cell viability was not significantly altered. These findings suggest that caution is required in the choice of certain lipid-based transfection reagents for gene silencing experiments, particularly when assessing cholesterol metabolism.

RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.

Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4%), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

Abstract LB-248: Development of lipid-based transfection reagents for efficient expression of transgenes in hard-to-transfect cell types

Abstract How to balance high efficiency of transgene expression with low cytotoxicity is a central concern for cell biology. Developing lipid-based transfection reagents that have those attributes and can be conveniently used for multiple cell types is critical for studies in cancer biology and cell biology. To address these concerns, ATCC has developed two lipid-based transfection reagents, GeneXPlus and TransfeX™ that provide a powerful tool for efficiently transfecting a broad spectrum of cell types including continuous cancer cell lines, stem cells, immortalized cells, and primary cells with little or no cytotoxicity. For recognized hard-to-transfect cancer cell lines, like THP-1, SH-SY5Y, and Raw 264.7 cells, we have achieved over 30% GFP expression without obvious cytotoxicity by using the GeneXPlus and an EF1α-GFP vector. TransfeX™ has been found to be highly suitable for the transfection of stem cells, immortalized cell lines, and many types of primary cells such as mammary, renal and bronchial epithelial cells, dermal microvascular endothelial cells, and human dermal fibroblasts. We have developed optimized transfection protocols for the TransfeX™ transfection reagent for different cell types and achieved over 50% transfection efficiency and low cytotoxicity. ATCC will provide data showing the broad spectrum of cell types successfully transfected with either GeneXPlus or TransfeX™. Citation Format: Dezhong Yin, Joy Wells, James Clinton, Lysa-Anne Volpe, Kevin Grady, Jeanmarie Curley. Development of lipid-based transfection reagents for efficient expression of transgenes in hard-to-transfect cell types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2014-LB-248