AP-4F,antennapedia peptide linked to an amphipathic alpha helical peptide,increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells

Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic alpha helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)-DNA typically yields transfection efficiencies of 35.4+/-3.3% with low levels of cell death (8.1+/-1.0%). Pre-treatment of the Lipofectamine 2000-GFP-DNA complexes with AP-4F for 5 min increased transfection to 58.2+/-2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10-20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin beta-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.     

An electroporation protocol for efficient DNA transfection in PC12 cells

A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

Differential behaviour of lipid based and polycation based gene transfer systems in transfecting primary human fibroblasts: a potential role of polylysine in nuclear transport.

DNA delivery systems for gene therapy applications have to be able to trigger the uptake of plasmid DNA into the nucleus. We have tested two types of non-viral vector systems, lipofection (cationic lipid-based, using Lipofectamine) and polyfection (cationic polymer-based, using glycerol enhanced transferrinfection), for their ability to transfect confluent, contact inhibited primary human fibroblasts. While both systems worked well with growing fibroblasts, polyfection was superior with confluent cells. A slight reduction in cell associated plasmid DNA was observed with resting cells, but it was similar for both types of complexes. Lipofectamine showed a prevalence for transfecting cycling cells as judged by costaining transfected cells with cell cycle markers. No such bias was observed when glycerol enhanced transferrinfection was used. Microinjection of plasmid DNA/polylysine complexes into the cytoplasm of fibroblasts resulted in a higher percentage of expressing cells than injection of plasmid DNA, offering an explanation for the higher transfection levels obtained with transferrinfection in non-growing cells.     

Differential behaviour of lipid based and polycation based gene transfer systems in transfecting primary human fibroblasts: a potential role of polylysine in nuclear transport

DNA delivery systems for gene therapy applications have to be able to trigger the uptake of plasmid DNA into the nucleus. We have tested two types of non-viral vector systems, lipofection (cationic lipid-based, using Lipofectamine) and polyfection (cationic polymer-based, using glycerol enhanced transferrinfection), for their ability to transfect confluent, contact inhibited primary human fibroblasts. While both systems worked well with growing fibroblasts, polyfection was superior with confluent cells. A slight reduction in cell associated plasmid DNA was observed with resting cells, but it was similar for both types of complexes. Lipofectamine showed a prevalence for transfecting cycling cells as judged by costaining transfected cells with cell cycle markers. No such bias was observed when glycerol enhanced transferrinfection was used. Microinjection of plasmid DNA/polylysine complexes into the cytoplasm of fibroblasts resulted in a higher percentage of expressing cells than injection of plasmid DNA, offering an explanation for the higher transfection levels obtained with transferrinfection in non-growing cells.     

Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Improved transfection efficiency of cultured human cells

We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine (Life Technologies) and Effectene (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5-3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.     

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.     

鸡原始生殖细胞转染条件优化

为获得鸡原始生殖细胞(primordial germ cells,PGCs)的最佳转染效率,本研究比较不同质粒用量和不同细胞数在3种转染试剂(Lipofectamine 2000、3000和LTX&Plus Reagent)中PGCs的转染效率,利用荧光激活细胞分选技术(fluorescence activated cell sorting technology,FACS)辅助优化Lipofectamine 3000转染试剂,经FACS进一步分选获得带绿色荧光蛋白(GFP)的PGCs,继续培养3周后,移植回注到受体鸡胚中,移植3.5 d后分离性腺拍照观察。结果显示,转染试剂Lipofectamine 3000的转染效率最高,质粒、Lipofectamine 3000转染试剂和PGCs细胞数的配比为3μg:4μL:0.5×10⁴个,转染5 h转染效率最高,达到23.4%,与现有的研究结果相比提高了2倍以上。移植回注PGCs到受体鸡胚中,荧光显微镜观察到鸡胚性腺中有GFP阳性细胞。本研究综合考虑转染试剂、质粒用量和细胞数量的影响因素以优化PGCs的转染条件,为高效制备转基因鸡及基因编辑鸡的研究奠定基础。     

Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO).

Electroporation is one of the most common methods used transform mammalian cells with plasmids. This method is versatile and can be adapted to meet the requirements of many cell lines. However, sometimes the efficiency of this method is low. We demonstrate that dimethyl sulfoxide (DMSO) facilitated a better DNA uptake in four different cell lines (HL60, TR146, Cos-7 and L132). The cells were electroporated with a beta-Gal expression plasmid in a medium containing DMSO (1.25%) during, and for 24 h after the pulse. In all these cells a dramatic (up to 8-fold) increase in transfection efficiency occurred after this treatment. This method opens up the possibility of using electroporation even in cells which are difficult to transfect.     

Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3)-fold and 1.5±0.7×10(3)-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p?=?0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Effectiveness of liposomes to transfect livestock fibroblasts

The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.     

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.