质粒DNA同源重组法构建腺病毒载体hosm

目的 构建含hosm基因的重组腺病毒载体,观察其对肿瘤细胞生长的影响。方法 用脂质体介导的粒DNA同源重组方法构建含人osm基因的重组缺陷型腺病毒载体,用PCR法鉴定所构建的ad—hosm载体。结果 成功构建了含hosm基因的重组腺病毒载体。结论 脂质体介导的质粒DNA同源重组法能有效构建重组腺病毒栽体;PCR法简化了阳性重组腺病毒的鉴定程序,为进一步研究重组腺病毒介导人osm基因对肿瘤细胞的生物学活性的影响提供了条件。     

阳离子脂质体对重组腺病毒转染效率的影响

目的　观察不同因素对重组腺病毒转染效率的影响。方法　以重组腺病毒去感染小鼠T淋巴瘤细胞株EL 4,加入不同的刺激因子和lipofectamine,通过计数X gal染色阳性细胞数测定转染效率。结果　rmIL 2 ,植物血凝素均不能改善重组腺病毒对EL 4的感染效率 ,而阳离子脂质体可以明显提高重组腺病毒对EL 4的感染效率。结论　可以利用阳离子脂质体来提高重组腺病毒的转染效率。     

A method to monitor DNA transfer during transfection

This report describes a method for monitoring the transfer of DNA during transfection. This method involves random labeling of plasmid DNA with fluorescein-12-dUTP, flow cytometric detection and sorting of the fluorescent transfected cells, and laser confocal fluorescence microscopic determination of the intracellular location of the plasmid DNA. By this method, >95% of the sorted cells were labeled, indicating a >95% specificity of the sorting procedure. The sorted cells were viable, as indicated by their ability to exclude trypan blue dye (>98% cells excluded the dye) and to maintain cell growth. The results of the kinetics of the Lipofectin transfection technology show that the fraction of the cells that internalized plasmid DNA increased from 10% at 1 hr after initiation of the transfection procedures to 18% at 3 hr. This method does not require protein expression, does not require the use of selection pressure such as drug treatment to isolate the cells that internalized DNA, and can be used to study the early events of DNA transfection.     

Ultrasound enhances the transfection of plasmid DNA by non-viral vectors

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.     

腺病毒载体的研究及应用进展

病毒载体能够携带外源基因并包装成感染性病毒颗粒,介导外源基因的转移和表达。腺病毒载体转导目的基因具有高效率、低致病性、高滴度以及在体内不整合入宿主细胞染色体等优点,被认为是最有效的转基因载体之一,哺乳动物来源的各种腺病毒被广泛用于基因治疗、肿瘤治疗及疫苗等领域。此文对腺病毒载体的特点及研究进展做一综述。     

Development of PEGylated adenovirus vector with targeting ligand

For effective gene therapy, a vector system that transduces the therapeutic gene into target cells efficiently and safely is essential. Adenovirus (Ad) vectors frequently are used for gene therapy research, especially cancer gene therapy, because of their high transduction efficiency. However, broad clinical utility of Ad vectors have not yet been achieved owing to problems related to several properties inherent to Ads. Systemic administration of Ad vectors leads to acute virus accumulation and undesirable transgene expression in the liver, with subsequent inefficient systemic cancer-targeted therapy and pronounced hepatotoxicity. Furthermore, most people have Ad-neutralizing antibodies, which hamper gene expression efficiency. Chemical conjugation of Ad surface with polyethylene glycol (PEG) (PEGylation) is one of the promising strategies to overcome these problems. Furthermore, PEGylation of Ad vectors with targeting ligands on the tip of PEG, which alter the transfection range of Ad vectors will improve the safety and efficiency of Ad gene-delivery vectors. In this review, we describe the molecular biology of Ads and outline this PEGylation approach including our data.     

Plasmid DNA and siRNA transfection of intestinal epithelial monolayers by electroporation

This study was conducted to evaluate the ability of electroporation to efficiently transfect differentiated intestinal epithelial monolayers with plasmid DNA and to determine whether electroporation can transfect these monolayers with short-interfering RNA (siRNA) to cause gene silencing. Confluent T84 monolayers were transfected with reporter plasmids expressing luciferase or green-fluorescent protein or with siRNA directed against the nuclear envelope proteins lamin A/C using electroporation. Optimized electroporation conditions resulted in luciferase and GFP expression. Both intracellular uptake of fluorescently labeled plasmid and expression of the reporter genes increased with increasing electroporation strength and DNA concentration. When monolayers were transfected by lipofection with the reporter plasmids, expression and DNA uptake were less than for electroporation. Electroporation was also found to transfect monolayers with siRNA, which resulted in up to 90% inhibition of targeted protein production. Silencing occurred within 24h of transfection and increased with increasing siRNA concentration. These results suggest that electroporation can provide a valuable research tool for transfection of intestinal epithelial monolayers and other differentiated cell systems, and may ultimately be useful for clinical gene therapy applications.     

Efficient transfection of DNA by liposomes formulated with cationic gemini amphiphiles

Cationic liposomes formulated with neutral 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cationic gemini surfactants were used for transfecting different cell lines with a reporter gene. The efficiency in the transfection has been correlated to the high extent of DNA condensation observed by circular dichroism, condensation shown to depend heavily on the gemini spacer structure. Transfection efficiency was better than that obtained with a commercial lipofection kit.     

Quantitative analysis of the leaky expression of adenovirus genes in cells transduced with a replication-incompetent adenovirus vector

Theoretically, adenovirus (Ad) genes should not be expressed following transduction with a replication-incompetent Ad vector because the E1A gene, which is essential for the expression of other viral gene, is deleted in a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with an E1-deleted Ad vector, leading to an induction of cellular immunity against Ad proteins. To date, no detailed analysis of the leaky expression profiles of Ad genes has been performed. In this study, we systematically examined the expression profiles of Ad genes in cells following transduction with a replication-incompetent Ad vector (Ad-L2) at multiplicities of infection (MOIs) of 10 and 100 using real-time RT-PCR. Significant expression was found for the E4 and pIX genes following transduction with Ad-L2 in cultured cells. The expression levels of the E4 and pIX genes were approximately 30- to 600-fold lower than those of the transgene (firefly luciferase), and 50- to 5000-fold lower than those of the E4 and pIX genes following transduction at the same MOI with the wild-type Ad. Unexpectedly, expression levels of the major capsid proteins were approximately the same as, or even slightly above, the background levels (Ad gene expression levels in mock-transduced cells). This study provides valuable information for the design of a safe and efficient replication-incompetent Ad vector.     

携带内皮抑素复制缺陷型腺病毒载体的构建及其应用

目的构建鼠源性内皮抑素(mEndostatin)重组腺病毒真核表达载体,并将其转染胰腺癌细胞株,探讨其体外抑制血管形成的活性。方法 以mEndostatin质粒为模板通过PCR方法扩增回收mEndostatin,将mEndostatin连接到腺病毒载体pCA13中,构建pCA13-mEndo表达质粒。将此表达质粒与腺病毒重组质粒pBGHE3通过lipofectamine 2 000共同转染293细胞,经同源重组产生重组腺病毒Ad-mEndo,用氯化铯密度梯度超速离心法纯化,用50％组织培养感染剂量法测定病毒滴度。体外转染胰腺癌细胞株AsPC-1、BxPC-3,并测定感染的胰腺癌细胞上清液mEndostatin的表达及观察其抑制血管形成的活性。结果 扩增得到的mEndostatin经酶切鉴定、PCR扩增、DNA测序证实为鼠源性内皮抑素,重组腺病毒Ad-mEndo的滴度为6.3×1010pfu／ml,体外能高效转染胰腺癌细胞株,可分泌表达有抑制血管形成活性的mEndostatin。结论本实验所构建的mEndostatin重组腺病毒表达载体能有效表达具有生物活性的mEndostatin,为体内胰腺癌的基因治疗奠定了基础。     

串联表达Fas shRNA腺病毒载体的构建

目的利用串联表达质粒pEGFP6-1-siFas1+siFas2和BDAdeno-X系统构建腺病毒表达Fas-shRNA载体。方法双酶切pEGFP6-1-siFas1+siFas2串联表达质粒和pShuttle2质粒,T4DNA连接酶连接胶回收片段,转化感受态Escherichiacoli(E.coli)DH5α菌株,挑取单克隆菌落LB(Luria-Bertani)培养基中扩增培养;小量质粒试剂盒提取pShuttle2-siFas1+siFas2质粒,酶切鉴定;双酶切pShuttle2-siFas1+siFas2质粒,T4DNA连接酶连接酶切产物和腺病毒BDAdeno-XViralDNA。回收连接产物,SwaI酶切去除未重组的腺病毒质粒,再回收酶切产物,转化感受态E.coliDH5α;聚合酶链反应(PCR)筛选鉴定阳性克隆;提取pAdeno-siFas1+siFas2质粒,进一步酶切鉴定;重组腺病毒pAdeno-siFas1+siFas2质粒线性化,经脂质体转染HEK293A细胞;收细胞进行裂解收病毒。结果构建表达2个Fas-shRNA的串联重组腺病毒载体pAdeno-siFas1+siFas2,经酶切鉴定和PCR分析,重组腺病毒质粒构建正确;经HEK293A细胞包装成功。结论成功构建表达2个Fas-shRNA的串联重组腺病毒载体pAdeno-siFas1+siFas2,为进一步转染细胞和感染小鼠抑制Fas基因表达奠定了基础。     

Impact of convective flow on the cellular uptake and transfection activity of lipoplex and adenovirus

An in vitro cell culture model that mimics in vivo extracellular environment would be useful in developing in vivo gene delivery system. In the present study, a parallel flow model was applied to investigate the impact of convective flow on cellular uptake and transfection activity in endothelial cells. LipofectAMINE PLUS and adenovirus were used as model vectors, which bind cells via electrostatic- and ligand-receptor interactions, respectively. Whereas a convective flow increased the total amount of vector passing through the flow chamber by 3 orders of magnitude, uptake was increased by less than 10-fold, suggesting that the flow severely inhibited cellular uptake by reducing the retention time in the chamber and/or by diminishing the affinity between the cell and vector. Moreover, the uptake of both vectors was increased in a shear stress-dependent manner to a comparable extent, suggesting that the effect of flow on the cellular uptake was not significant. In contrast, transfection efficiency (TE), expressed as the transfection activity normalized by the cellular uptake of vectors was dramatically stimulated by shear stress, only when LipofectAMINE PLUS was used. Since the activities of the CMV promoter were unaffected by a shear stress, it is possible that altered intracellular trafficking may responsible for the improvement in lipoplex-mediated TE, presumably related to the cellular uptake pathway.     

Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification

Abstract

Context: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications.

Objective: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells.

Materials and methods: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay.

Results and discussion: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000? showed decreased cell viability.

Conclusion: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.     

Improved transfection efficiency of CS/DNA complex by co-transfected chitosanase gene

Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatuscsn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by beta-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.     

Optimization of renal transfection using a renal suction-mediated transfection method in mice

Background: We previously developed a suction-mediated transfection method in mice.

Purpose: The purpose of this study was to optimize the suction-mediated transfection conditions using a pressure-controlled computer system for efficient and safe kidney-targeted gene delivery in mice.

Methods: Naked pCMV-Luc was injected into the tail vein in mice, and then the right kidney was suctioned by a device of the suction pressure-controlled system. The effects of renal transfection conditions, such as the suction pressure degree, suction pressure waveform and device area were evaluated by measuring luciferase expression. In addition, renal injury was examined.

Results: The renal suction-mediated transfection method at ?30?kPa showed high transgene expression. The renal suction waveform did not affect the transfection activity. Under the optimized conditions, the high transgene expression was mostly observed at the renal suctioned site. The transfection conditions used did not induce histological defects or increases in two renal injury biomarkers (Kidney injury molecule-1 mRNA and Clusterin mRNA).

Discussion and conclusion: We have clarified the transfection conditions for efficient and safe transfection in the kidney using the suction-mediated transfection method in mice.     

腺病毒及紫杉醇阴离子脂质体共传递复合物的制备及体外细胞转染

目的 制备腺病毒及紫杉醇阴离子脂质体(AL-Ad5-PTX)共传递复合物,考察该复合物物理性质及体外细胞转染效率.方法 先后用薄膜超声法和钙离子融合法制得AL-Ad5-PTX;测定AL-Ad5-PTX中紫杉醇的含量;测定粒径及电位;用透射电镜观察其形态;以表达荧光素酶的重组腺病毒作为病毒载体,比较AL-Ad5-PTX和裸腺病毒(naked Ad5)在富含柯萨奇腺病毒受体(CAR)或缺乏CAR受体细胞中的转染效率.结果 AL-AdS-PTX中紫杉醇包封率为82.70％±2.47％,粒径为241.6 ±2.6 nm,粒径分布为0.196±0.006,电位为-50.4±5.4 mV.AL-Ad5-PTX在两种细胞中的转染效率高于naked Ad5(P＜0.05).结论 成功制备了能够同时转运腺病毒及紫杉醇的复合物AL-Ad5-PTX,能够提高naked Ad5在细胞中的转染效率;HPLC法检测AL-Ad5-PTX中紫杉醇含量,操作简便、准确、可靠.     

介绍一种改量的粪便细菌基因组DNA提取试验方法

目的 观察改变细菌裂解温度对提取粪便细菌基因组DNA量和纯度的影响.方法 收集10例肝硬化患者粪便,每例患者粪便等分成两份,再随机分成两组.采用QIAamp DNA Stool Mini Kit试剂盒（国际通用粪便细菌基因组DNA提取试剂盒）进行抽提,一组（A组）按照试剂盒说明书温度要求进行粪便细菌基因组DNA提取,一组（B组）对试剂盒细菌裂解温度改良后再进行提取.结果 与按照试剂盒说明书温度进行提取组相比,改变细菌裂解温度后粪便基因组DNA提取量明显增高（P〈0.05）,而粪便基因组DNA提取纯度（OD260/280、OD260/230）无明显变化（P〉0.05）.结论 改变细菌裂解温度能明显提高粪便细菌基因组DNA提取量,而不改变粪便细菌基因组DNA的提取纯度.该方法可以解决因粪便采样量少导致粪便基因组DNA提取量不足的问题.     

Ultrasound enhancement of in vitro transfection of plasmid DNA by a cationized gelatin

In vitro transfection efficiency of a plasmid DNA for rat gastric mucosal (RGM)-1 cells was enhanced by ultrasound (US) irradiation. Ethylenediamine was introduced to the carboxyl groups of gelatin to prepare a cationized gelatin as the vector of plasmid DNA encoding luciferase. An electrophoresis experiment revealed that the cationized gelatin was mixed with plasmid DNA at the weight ratio of 5.0 to form a cationized gelatin-plasmid DNA complex. The complex obtained was about 200nm in diameter with a positive charge. When incubated with the cationized gelatin-plasmid DNA complex and subsequently exposed to US, RGM-1 cells exhibited a significantly enhanced luciferase activity although the extent increased with an increase in the DNA concentration, in contrast to the cationized gelatin alone with or without US irradiation and US irradiation alone. US irradiation was also effective in enhancing the activity by free plasmid DNA although the extent was less than that of the complex. The US-induced enhancement of luciferase activity was influenced by the exposure time period, frequency, and intensity of US. The activity enhancement became higher to be significant at the irradiation time period of 60 s and thereafter decreased. A series of cytotoxicity experiments revealed that an increase in the irradiation time period and intensity of US decreased the viability of cells themselves. It is possible that US irradiation under an appropriate condition enables cells to accelerate the permeation of the cationized gelatin-plasmid DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. These findings clearly indicate that US exposure is a simple and promising method to enhance the gene expression of plasmid DNA.     

Kinetic analysis of protein production after DNA transfection

The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8x10(4)- and 4x10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection相似文献   

Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.