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,EL转染试剂在Eca-109细胞株中的转染效果最好,在另外两种细胞中转染效果不佳。在Eca-109细胞株中,转染效果最佳检测时间为转染36 h时,细胞存活情况不受转染试剂影响。EL转染试剂与Hsa-miR-6743质粒最佳转染比例范围为1:4~1:2。EL转染试剂的转染效果受细胞种类、转染试剂与质粒比值以及转染时间的影响。     

Current standards and pitfalls associated with the transfection of primary fibroblast cells

Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.     

Systematics of naviculoid diatoms (Bacillariophyta): A preliminary analysis of protoplast and frustule characters for family and order level classification

The relationships between 49 naviculoid diatoms, currently arranged in 14 families and four orders were investigated using cladistic analysis in order to test the types of characters used in diatom systematics and to assess how well the current classification reflects possible phylogenetic relationships in this group. Some of the families and orders comprise taxa with different protoplast characters, or taxa with similar protoplast arrangements are placed in separate families or orders. Therefore as both cell wall and protoplast characters were used, three analyses were undertaken; total data, protoplast data and frustule data. The analyses support the recognition of the Mastogloiales (unequivocally) and the Cymbellales (largely) but indicate that some of the familial groupings are more ambiguous. The members of the Berkeleyaceae, Berkeleya, Parlibellus and Climaconeis, were never grouped together and Achnanthes brevipes never grouped with the other monoraphid diatoms, but usually with members of the Mastogloiales (total and protoplast data). Similarly, Round et al.’s familial groupings within the Cymbellales do not emerge from our analyses. Our results support the hypothesis that monoraphid genera have arisen independently from different naviculoid diatoms, and that Achnanthes sensu stricto should be transferred to the Mastogloiales. Some of the problems associated with incomplete information and inaccurate terminology are discussed briefly.     

Synthesis of Novel 5′-Uridine-Head Amphiphiles as Model for DNA Molecular Recognition

Here we describe uridine functionalization in the 5′ position, which provides new classes of cationic and nonionic amphiphiles specifically designed as DNA transfection agents. The synthetic procedures developed to obtain the cationic uridine-head surfactants prevented intramolecular cyclization that occurs when uridine is functionalized in this position without using protecting groups in the uracil.     

Importin‐7 Mediates Nuclear Trafficking of DNA in Mammalian Cells

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy‐dependent way. The imp7‐dependent pathway was specifically competed by excess DNA but not by excess of maltose‐binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly‐l ‐lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non‐redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response .     

Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.     

Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.

The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

基因组重排选育胸苷磷酸化酶高产菌株

以短乳杆菌为研究对象,通过基因组重排技术选育胸苷磷酸化酶高产菌株。首先采用紫外复合诱变筛选出EA42、EB27作为基因组重排育种的亲本并制备成原生质体,分别采用紫外照射50min和60℃水浴加热60min双亲灭活原生质体,然后用质量分数40％PEG6000,30℃恒温诱导融合10min进行基因组重排。经过3轮基因组重排育种,成功选育出3株胸苷磷酸化酶高产菌株,其中菌株F3-36在菌体发酵量提高的前提下,进行5次传代测试其胸苷磷酸化酶活均在2．500U／mg湿菌体,比原始菌株酶活提高了260％。