Monte Carlo calculations of energy deposition distributions of electrons below 20 keV in protein

The distributions of energy depositions of electrons in semi-infinite bulk protein and the radial dose distributions of point-isotropic mono-energetic electron sources [i.e., the so-called dose point kernel (DPK)] in protein have been systematically calculated in the energy range below 20 keV, based on Monte Carlo methods. The ranges of electrons have been evaluated by extrapolating two calculated distributions, respectively, and the evaluated ranges of electrons are compared with the electron mean path length in protein which has been calculated by using electron inelastic cross sections described in this work in the continuous-slowing-down approximation. It has been found that for a given energy, the electron mean path length is smaller than the electron range evaluated from DPK, but it is large compared to the electron range obtained from the energy deposition distributions of electrons in semi-infinite bulk protein. The energy dependences of the extrapolated electron ranges based on the two investigated distributions are given, respectively, in a power-law form. In addition, the DPK in protein has also been compared with that in liquid water. An evident difference between the two DPKs is observed. The calculations presented in this work may be useful in studies of radiation effects on proteins.     

Gene delivery into hepatocytes with the preS/liposome/DNA system

Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system.     

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol-DOPE/DNA lipoplexes in serum

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.     

Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.     

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches (“flipids”) that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.     

Cationic micelle: A promising nanocarrier for gene delivery with high transfection efficiency

Micelles have demonstrated an excellent ability to deliver several different types of therapeutic agents, including chemotherapy drugs, proteins, small‐interfering RNA and DNA, into tumor cells. Cationic micelles, comprising self‐assemblies of amphiphilic cationic polymers, have exhibited tremendous promise with respect to the delivery of therapy genes and gene transfection. To date, research in the field has focused on achieving an enhanced stability of the micellar assembly, prolonged circulation times and controlled release of the gene. This review focuses on the micelles as a nanosized carrier system for gene delivery, the system‐related modifications for cytoplasm release, stability and biocompatibility, and clinic trials. In accordance with the development of synthetic chemistry and self‐assembly technology, the structures and functionalities of micelles can be precisely controlled, and hence the synthetic micelles not only efficiently condense DNA, but also facilitate DNA endocytosis, endosomal escape, DNA uptake and nuclear transport, resulting in a comparable gene transfection of virus.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGNE 6 transfection technology. The nocovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a mximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G₂/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.     

Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA

Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector.     

阳离子脂质体转染人类骨骼肌原代细胞的初步研究

探讨不同脂质体介导基因转染人类骨骼肌原代细胞的转染效率和基因的表达.将含有β-半乳糖苷酶LacZ结构基因的质粒,用三种不同的阳离子脂质体导入人类骨骼肌原代细胞中,通过X-Gal染色观察不同的转染效率.结果发现,Fugene 6转染效率最高,蓝染细胞达10%,其脂质体与DNA的最佳比例为3∶ 2.Fugene 6可有效地将外源基因导入骨骼肌原代细胞,而且外源基因可以长效高效地表达,有望用来作为基因治疗的载体.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Enhanced gene transfer into brain capillary endothelial cells using Antp-modified DNA-loaded nanoparticles

Brain capillary endothelial cells (BCECs) have been considered as one of the primary targets for cerebral gene therapy. However, the cells, well-known for their poor function of endocytosis, are difficult to be transfected by general non-viral vectors. The aim of this study was to enhance the efficiency of transfection and expression in BCECs of DNA/polymer nanoparticles with the modification of membrane-penetrating peptide, Antennapedia peptide (Antp) polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare Antp-modified DNA-loaded nanoparticles with a complex coacervation technique. After a 20-min transfection, the efficiency, in terms of transfection and expression, of DNA/PEI NP or DNA/PAMAM NP was enhanced significantly with the modification of Antp. After a 3-h transfection of DNA/Antp/PEI NP, there was no difference in cellular uptake but an enhancement in gene expression, compared to DNA/PEI NP alone. However, both the transfection and expression efficiency of DNA/PAMAM NP were enhanced using Antp. These observations suggest that Antp can increase the membrane-penetrating ability of DNA-loaded nanoparticles, which can be employed as novel non-viral gene vectors.     

Synthesis of galactosyl compounds for targeted gene delivery

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.     

Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine

Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.     

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.     

聚乙烯亚胺转基因影响因素的测定及其优化

聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .     

Impact of BTG2 expression on proliferation and invasion of gastric cancer cells in vitro

BTG2 (B cell translocation gene 2) is downregulated in several human tumors and has been known as a tumor suppressor in carcinogenesis of thymus, prostate, kidney, and liver. However, little is known about the role BTG2 plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of BTG2 on the growth, proliferation, apoptosis, invasion and cell cycle of the gastric cancer cell lines SGC7901 and MKN45. BTG2 cDNA was insected into a constitutive vector pcDNA3.1 followed by transfection in gastric cancer cell line MKN45 and SGC7901 by using liposome. Then stable transfectants were selected and appraised. The apoptosis and cell cycles of these transfectants were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay, respectively. The invasion of these clones was analyzed by using cell migration assay. MKN-BTG2 (MKN45 with stable transfection of BTG2 gene) and SGC-BTG2 (SGC7901 with stable transfection of BTG2 gene) grew slower than their control groups, respectively. The cell counts of MKN-BTG2 in the fourth, fifth, sixth and seventh days were significantly fewer than those of control groups (P < 0.05). Those of SGC-BTG2 in the fourth fifth, sixth and seventh days were significantly fewer than those of control groups too (P < 0.05). Cell cycle analysis showed that proportions of MKN-BTG2 and SGC-BTG2 cells in G0–G1 and S were different significantly with those of their control groups, respectively (P < 0.05). The apoptosis rate of MKN-BTG2 was significantly higher than those of control groups (P < 0.05). Results of colony-forming assay showed that the colon formation rates of MKN-BTG2 and SGC-BTG2 were lower than those of their control groups (P < 0.05). The results of cell migration assay showed that the cell migration rates of MKN-BTG2 and SGC-BTG2 were not significantly different with those of their control groups (P > 0.05). BTG2 can restrain the growth and proliferation of gastric cancer cells powerfully. It can reduce some malignant phenotype of these tumor cells. But it could not impact the ability of invasion of gastric cancer cells, so could not restrain the metastasis of gastric cancer. In gastric cancer, BTG2 could be thought as a tumor-inhibiting gene in some distance, so the gene could be a potential target of gene therapy.     

新城疫病毒7793 HN蛋白在昆虫SF9细胞中的表达研究

本实验对新城疫病毒(newcastle disease virus,NDV) 7793 HN蛋白在杆状病毒表达系统中表达进行研究。提取病毒RNA并将其逆转录成cDNA,经PCR同义点突变在HN基因片段中加入NcoⅠ/XhoⅠ酶切位点,通过该酶切位点将HN基因克隆至穿梭载体p FastBac1质粒以构建重组质粒pFastBac1HTB-HN,然后用脂质体转染pFastBac1HTB-HN杆粒至昆虫SF9细胞。28℃无菌培养含pFastBac1HTB-HN杆粒的SF9细胞48 h,收集细胞培养上清液中的第一代病毒,感染SF9细胞,置28℃无菌培养60 h,收集细胞培养上清液,离心去除细胞碎片,取上清液中的第二代病毒,继续感染SF9细胞,置28℃无菌培养72 h,收集SF9细胞,用SDSPAGE和Western blotting验证HN蛋白的表达。SDS-PAGE和Western blotting均显示HN杆粒感染的SF9细胞成功表达了HN蛋白。本研究结果为进一步研究NDV7793-HN蛋白的抗肿瘤作用提供可靠试验依据。     

牛αS1酪蛋白5′调控序列驱动报告基因 LacZ 在奶山羊乳腺上皮细胞中的表达

αS1酪蛋白是牛乳中含量最高的酪蛋白.本研究克隆了牛αS1酪蛋白5′调控序列约1.2 kb的片段,应用基因重组技术将其插入 lacZ 基因上游,构建真核表达载体gαs1p psv.胶原酶消化法对奶山羊乳腺上皮细胞进行了分离培养,并用免疫荧光法鉴定了乳腺上皮细胞. 将重组载体转染奶山羊乳腺上皮细胞,在细胞培养液中,转染后24 h可以检测到 lacZ 基因的表达,之后表达水平有逐渐升高的趋势,72 h开始降低,144 h降到最低;在细胞破碎液中,转染后24 h表达水平最高,之后表达水平有逐渐降低的趋势,144 h降到最低.转染细胞的第1代表达水平最高,随细胞传代表达水平降低,传到第3代时基本检测不到表达产物. 对转染后的细胞进行了β 半乳糖苷酶原位细胞染色. 实验证明,得到的牛αS1酪蛋白5′调控序列能指导外源基因在奶山羊乳腺上皮细胞中表达.     

Drosophila cells and ecdysterone: a model system for gene regulation

When Drosophila cell lines are exposed to physiological doses of the steroid molting hormone, ecdysterone, they enter mitotic arrest and differentiate morphologically. These responses are accompanied by specific changes in gene expression. Several enzyme activities (acetylcholinesterase, beta-galactosidase, dopa decarboxylase, and catalase) are induced and the synthesis of a cytoplasmic actin and the four small heat-shock proteins is initiated. Several of these ecdysterone inducible genes have been physically isolated and characterized, in several cases by DNA sequencing. Current studies focus on introducing cloned ecdysterone inducible genes into responsive cells by DNA mediated transfection. Once it is clear that these introduced genes acquire the normal pattern of hormone-regulated gene expression in the cell, in vitro mutagenesis can be used before transfection to modify their structure. Transient expression, then, can become a functional assay to define regions of DNA flanking the coding region of inducible genes that are needed for proper gene expression and regulation in cultured cells.