EL转染试剂转染食管鳞癌细胞的条件优化

该文探讨了关于EL转染试剂转染Hsa-miR-6743质粒至食管鳞癌细胞转染效果的影响因素.以食管鳞癌细胞株Eca-109、TE-1和Eca-9706为研究对象,GFP标记的Hsa-miR-6743为报告基因,通过倒置荧光显微镜检测荧光信号优化转染试剂和质粒比值.结果表明,食管鳞癌细胞的种类影响EL转染试剂的转染效果,...     

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays.     

Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells.     

Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.     

Transfection of embryonal carcinoma cells at high efficiency using liposome-mediated transfection

Embryonal carcinoma (EC) cells are recognized as an excellent model system for studying the early stages of mammalian development. Many studies performed with EC cells involve transient transfection with promoter/reporter gene constructs and/or mammalian expression vectors. One of the limitations of working with EC cells is their inability to be transfected at high efficiency. In most cases, EC cells are transfected using the calcium phosphate method. The objective of this study was to identify protocols and culture conditions that significantly increase the transfection efficiency of EC cells. F9 EC cells were used for this purpose, because they are the EC cell line studied most commonly. We show that the transfection efficiency of F9 EC cells using the calcium phosphate method is less than 5%; whereas, their transfection efficiency can be improved approximately 15-fold using optimized culture conditions and liposome-based transfection reagents. Specifically, we demonstrate that more than 50% of F9 EC cells can be transfected using LipofectAMINE 2000. In addition to higher levels of transfection, there is much less plate-to-plate variation with liposome-based reagents as compared to transfection with calcium phosphate. Interestingly, transfection efficiency using these reagents was found to be inversely related to cell density. This contrasts sharply with the recommendation that transfection with LipofectAMINE 2000 or LipofectAMINE in conjunction with the PLUS reagent be performed at high cell densities. Given the improvements in transfection efficiency reported here, it will now be possible to perform studies with F9 EC cells that require transfection at significantly higher levels than that achieved using the calcium phosphate method. Overall, the highest transfection efficiencies were consistently obtained using LipofectAMINE 2000.     

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5h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...     

Gene delivery for genetically engineered mucosal cells with enhanced function

A non-viral transfection method for oral mucosal cells was investigated using a modified transfection method and five commercial transfection reagents. The CellFECTIN^TM gave the highest expression of a transfected gene. When the mucosal cells were transfected with 0.3 ng DNA/cell, the transfection efficiency was optimal, and the production of a reporter protein increased up to ten times higher than those with the other transfection reagents.     

Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications

Non-viral transfection is a promising technique that could be used to increase the therapeutic potential of stem cells. The purpose of this study was to explore practical culture parameters of relevance in potential human mesenchymal stem cell (hMSC) clinical and tissue engineering applications, including type of polycationic transfection reagent, N/P ratio and dose of polycation/pDNA polyplexes, cell passage number, cell density and cell proliferation. The non-viral transfection efficiency was significantly influenced by N/P ratio, polyplex dose, cell density and cell passage number. hMSC culture conditions that inhibited cell division also decreased transfection efficiency, suggesting that strategies to promote hMSC proliferation may be useful to enhance transfection efficiency in future tissue engineering studies. Non-viral transfection treatments influenced hMSC phenotype, including the expression level of the hMSC marker CD105 and the ability of hMSCs to differentiate down the osteogenic and adipogenic lineages. The parameters found here to promote hMSC transfection efficiency, minimize toxicity and influence hMSC phenotype may be instructive in future non-viral transfection studies and tissue engineering applications.     

Effect of osmolarity and presence of serum on the efficiency of cell transfection using immunoporation.

Immunoporation is a novel method of cell transfection based upon the use of a new type of beads, Immunofect beads, that can be targeted to make holes in different types of cells depending on the type of bead used. It is known that the efficiency of transfection of cells by some techniques can be affected by the presence of serum and another important factor that appears to affect transfection efficiency and cell viability is the osmolarity of the transfection medium. This report presents studies on the effects of serum and varying osmolarity on the efficiency of transfection using immunoporation. The results clearly indicate that in hypertonic media the presence of serum decreases the efficiency of transfection. In the case of osmolarity, increasing the osmolarity of the immunoporation medium increases the efficiency of transfection but above about 650 mOsm this increasing efficiency is offset by the much lower viability of the cells.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Enhancement and optimization of plasmid expression in femtosecond optical transfection

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non‐invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3‐fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO‐K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ～70% without compromising the transfection efficiency. Also, we report for the first time the successful photo‐transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Variation in enzymatic transient gene expression assays

We examined causes for high variability in data from enzymatic transient gene expression assays. Our results strongly suggest that variation in transfection efficiency is the major cause of data variation and can seriously compromise valid interpretation of data. We compared averaging data from multiple transfections and cotransfection of a second reporter gene as methods for correcting for variation in transfection efficiency. We found that transfection efficiency can be so highly variable that neither method necessarily overcomes the resulting bias in data. Depending upon the degree in variation in transfection efficiency, a combination of the two methods may be advisable. The need to normalize data for transfection efficiency is dependent upon the difference in strengths of promoters being tested and the relative variability of the transfection method used. We also show that the level of reporter gene expression between transfection experiments performed on different days can vary by more than 10-fold.     

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.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

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。     

Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.     

Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications

Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome-DNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs.