位点特异整合微环DNA的体内制备

目的:应用诱导表达的LR克隆酶系统,建立一种在细菌体内获得基于链霉菌噬菌体ФC31整合酶系统的位点特异整合型微环DNA的方法,为实现无细菌骨架等冗余序列的转基因奠定基础。方法:构建包含阿拉伯糖启动子的LR克隆酶系统和ФC31整合酶系统的亲本质粒,在L-阿拉伯糖的诱导下重组产生表达ФC31整合酶的微质粒和包含有目的基因和attB位点等元件的微环DNA。以限制性内切核酸酶酶切电泳定性其重组效率,qPCR定量分析微环、微质粒及亲本质粒的比例,定量计算重组效率。观察随着诱导时间的推进微环/微质粒值的变化。结果:细菌体内LR克隆酶系统可有效催化亲本质粒的重组,重组率达85%以上。相比商品化LR克隆酶体外反应具有更高的稳定性而且更经济。结论:获得了一种高效、稳定的细菌体内产生位点特异性整合型微环DNA的亲本质粒。     

聚乙烯亚胺包裹小环DNA形成的纳米颗粒传输外源基因的性质表征

聚乙烯亚胺(PEI)是一种具有良好生物安全性和生物相容性的非病毒载体,能高效转染肿瘤细胞。小环DNA是一种去除质粒细菌骨架,只含有目的基因表达框的环状DNA分子。与普通质粒相比,小环DNA具有表达效率高、持续时间长的优势。使用PEI包裹携带报告基因gfp和抑癌基因pten小环DNA载体,并利用各种技术手段分析了该传输系统的理化性质和生物学效应。凝胶阻滞实验、电镜实验及MTT实验分析结果表明利用PEI包裹小环DNA和质粒DNA体系性质无显著的差别,并且2种复合物对细胞毒性亦无明显差别;但是动态光散射实验结果显示由于PEI可以包裹更多数量的小环DNA,所以PEI包裹小环DNA形成的复合物粒径要略大于包裹质粒DNA形成的复合物粒径。荧光显微镜实验、real-time PCR分析和Western blotting分析结果表明,PEI包裹小环DNA形成的复合物对细胞的转染效率要远远高于PEI包裹质粒DNA所形成的复合物,并且小环所携带的外源基因的表达效率要远远高于质粒DNA所携带的外源基因的表达效率。实验结果表明,PEI包裹小环DNA形成的纳米颗粒在细胞转染过程中具有很高的表达效率,这一研究结果为PEI包裹小环DNA的非病毒载体系统在传输外源基因过程中的应用提供理论基础和技术支持。     

新型的DNA序列测定策略:PCR产物直接测序

DNA序列的测定在现代分子生物学中的应用越来越广泛,传统的模板制备方法是将待测序列的DNA片段插入质粒或病毒载体中,然后让其在宿主细菌细胞中增殖.尽管这套方法已被简化和标准化,但由于其涉及到活细胞的保存和使用,不可避免地会带来诸如载体和宿主细胞基因发生突变等不利影响.     

Stathmin SiRNA表达载体抑制stathmin的表达

目的:构建stathmin特异性SiRNA质粒表达载体,探讨其对鼻咽癌5-8F细胞stathmin的沉默作用.方法:合成用于stathmin基因特异性干扰表达的DNA片段,经退火形成双链DNA片段,片段克隆到质粒表达载体pGenesil 1.1上.载体导入JM109菌株进行筛选与扩增,采用酶切和测序对克隆表达载体进行鉴定.应用脂质体将鉴定后的重组表达质粒载体转入鼻咽癌5 -8F细胞,RT-PCR与Western Blot分析stathmin基因表达.结果:经酶切和测序鉴定,插入SiRNA质粒表达载体的stathmin特异性碱基序列和方向正确.重组质粒表达载体转染鼻咽癌细胞后,细胞转染效率达78.8 ±6.8％,stathmin基因在鼻咽癌中的表达明显下降.结论:构建的stathmin基因SiRNA质粒表达载体能抑制stathmin的表达.     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

靶向端粒酶逆转录酶（hTERT）RNAi载体的构建及活性评价

RNA干涉是由与特定基因同源互补的双链RNA,在体内以序列特异的方式引发靶基因的mRNA降解,从而导致转录后基因沉默的过程.为研究内源性的siRNA对靶基因的抑制效果,用pPUR/U6载体构建用于细胞内转录靶向hTERT基因的短发夹状siRNA表达质粒,并评价其对hTERT基因的抑制效果.将hTERT cDNA 3 565～3 583一段19 bp的DNA序列及其反向重复序列,用9 bp的连接序列连接后再接上5个T碱基,将此段DNA序列克隆至pPUR/U6载体U6启动子的下游,形成能在体内合成hTERT特异性短发夹状RNA的重组质粒载体pPUR/U6/hTERT.将pPUR/U6/hTERT与对照质粒pPUR/U6分别转染HepG2细胞.采用嘌呤霉素筛选和富集转染具有抗性的细胞.收集存活的细胞接种12孔板、提取RNA和蛋白质.以细胞计数法测定细胞的生长速度,RT-PCR和蛋白质印迹分析hTERT基因的表达,端粒重复放大测定法检测端粒酶活性,蛋白质印迹检测p53蛋白水平.与转染对照质粒pPUR/U6的细胞相比,转染pPUR/U6/hTERT的细胞其hTERT基因表达水平显著下降,端粒酶活性降低,细胞生长速度变慢,p53蛋白表达明显升高.以上结果表明,以DNA质粒为载体产生的内源性短发夹状siRNA能高效抑制hTERT基因的表达,有望成为基因功能研究的有力工具.     

医药其它

编码HIV病毒一l或HIV病毒一2蛋白质的DNA序列是新的.含上述新序列的表达载体质粒也是新的.这些新的表达载体质粒转化大肠杆菌细胞后能在其中表达.这     

AAV-ITR单链DNA微载体在小鼠骨骼肌中的表达

AAV-ITR单链DNA微载体是一种基于腺相关病毒(AAV)倒置末端重复序列(ITR)的基因表达载体(AAV-ITR ss DNA mini vector)。前期研究已证明AAV-ITR单链DNA微载体在HEK 293T细胞中具有较高的转染、表达效率。本文中将相同拷贝数的AAV-ITR单链DNA微载体、3?-ITR末端错配的AAV-ITR单链DNA微载体(AAV-ITRmm ss DNA mutant vector)、AAV-ITR双链DNA和质粒分别用Turbo Fect转入小鼠骨骼肌中,比较检测AAV-ITR单链DNA微载体与其他基因表达载体在小鼠体内1周、1个月及3个月的表达效率。组织切片经荧光显微镜观察及荧光灰度值分析表明,相同分子摩尔数的AAV-ITR单链DNA微载体比AAV-ITR双链DNA和质粒在不同时期表达效率都要高且更稳定。提取注射3个月后的肌肉组织的DNA,用荧光定量PCR分析比较各载体的存留分子数。RT-PCR的结果显示AAV-ITR单链DNA微载体在注射3个月后的存留分子数较其他载体高。综合结果显示AAV-ITR单链DNA微载体在动物体内具有表达效率高和长久稳定的优势,有可能开发为基因治疗的一种高效、稳定的新型载体。     

小鼠白细胞介素4基因的化学合成、克隆与表达

利用381A型DNA合成仪,分29个寡聚核苷酸片段化学合成了小鼠IL-4全基因,共442bp。以pUC12质粒作为载体,将所有合成片段分前后两组进行磷酸化、退火、连接和克隆,经过菌落原位杂交、酶切鉴定和质粒DNA序列分析,分别得到了含有小鼠IL-4前后两半基因片段的两种重组质粒,回收前半基因片段,插入到含有后半基因重组质粒的EcoRI和PstI酶切位点之间,成功地得到了含有小鼠IL-4全基因的重组质粒pFR101。将全合成基因插入到质粒pSM53中,得表达质粒pFR105,转化大肠杆菌TAP106,根据IL-4对CTLL细胞的作用,肯定了TAP106(pFR105)细菌中有小鼠IL-4活性蛋白的表达。     

肉葡萄球菌tat-gfp融合基因的构建与表达

将PCR扩增的肉葡萄球菌铁离子过氧化物酶基因(fepB)的双精氨酸转运(Tat)信号肽DNA序列与绿色荧光蛋白基因(gfp)亚克隆到pCX9质粒中构建可表达的tat-gfp融合基因,再将形成的pCX 19-Tat-GFP质粒电转化转入肉葡萄球菌宿主中.提取阳性克隆子质粒进行酶切验证,表明表达载体pCX 19-Tat-G...     

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.     

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non‐covalent complexation and intracellular delivery

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa‐arginine (R₈) and CHK₆HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep‐3, flanked with CH₃ on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide–plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non‐covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide–plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

质粒pAAV-HRE-CD151的构建和鉴定

目的为了增加CD151基因转染大鼠缺血心肌的效率和靶向特异性。方法以质粒pAAV-CD151为模板,采用克隆技术,构建一个含缺氧反应元件(hypoxia response element,HRE)和CD151基因序列的腺相关病毒载体,通过HRE促进CD151在大鼠缺血心肌的表达。结果经过测序鉴定成功构建了含缺氧反应元件的质粒pAAV-HRE-CD151。结论成功构建了pAAV-HRE-CD151质粒,为CD151治疗缺血性心血管病的靶向研究奠定了基础。     

Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes

Background

Peptide/DNA complexes have great potential as non-viral methods for gene delivery. Despite promising results for peptide-mediated gene delivery technology, an effective systemic peptide-based gene delivery system has not yet been developed.

Methods

This study used pCMV-Luc as a model gene to investigate the biodistribution and the in vivo efficacy of arginine peptide-mediated gene delivery by polymerase chain reaction (PCR).

Results

Plasmid DNA was detected in all organs tested 1 h after intraperitoneal administration of arginine/DNA complexes, indicating that the arginine/DNA complexes disseminated widely through the body. The plasmid was primarily detected in the spleen, kidney, and diaphragm 24 h post administration. The mRNA expression of plasmid DNA was noted in the spleen, kidney, and diaphragm for up to 2 weeks, and in the other major organs, for at least 1 week. Blood clearance studies showed that injected DNA was found in the blood as long as 6 h after injection.

Conclusions

Taken together, our results demonstrated that arginine/DNA complexes are stable in blood and are effective for in vivo gene delivery. These findings suggest that intraperitoneal administration of arginine/DNA complexes is a promising tool in gene therapy.     

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.     

Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle

Gene transfer into muscle upon injection of plasmid DNA is feasible but occurs with low frequency. However, by using electroporation after injection of plasmid DNA into mouse muscle it has been demonstrated that gene expression can be increased more than 150-fold. In this communication, we have used this technique in combination with plasmids containing a tandem repeat of three 72-bp DNA elements from the SV40 enhancer to study gene expression. Our results show that the combination of electroporation and a plasmid vector carrying these DNA elements results in increased and more persistent gene expression of the luciferase reporter gene in BALB/c mouse muscle. At 14 days after gene delivery, the gene expression was 16-fold higher in muscles injected and electroporated with the plasmid carrying the SV40 enhancers than with control plasmid. We have also studied the effects of the vehicle in which the plasmid was delivered, and the DNase inhibitor aurintricarboxylic acid (ATA), on gene expression. By combining ATA with 150 mM sodium phosphate buffer we were able to obtain a 2-fold increase in gene expression compared to delivery of the plasmid in physiological saline. These results are of importance for the development of efficient delivery techniques for naked DNA.     

Fast and accurate method for quantitating E. coli host-cell DNA contamination in plasmid DNA preparations

Plasmid DNA is being used successfully as a gene delivery vector in a variety of clinical applications. Similar to other pharmaceutical products for clinical use, the plasmid vectors must meet rigorous purity standards. One important contaminant is the DNA of the host cell used to produce the plasmids. We have developed a new method to accurately quantitate E. coli host-cell DNA in plasmid preparations. This method is based on kinetic PCR using the ABI PRISM 7700 with 23S rDNA as a target. This precise assay is significantly faster and has a lower limit of quantitation than the currently used Southern-based methods.     

Animal-free production of ccc-supercoiled plasmids for research and clinical applications

The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application. Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances. Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms.     

Therapeutic dendritic cell vaccine preparation using tumor RNA transfection: A promising approach for the treatment of prostate cancer

Background

Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

Methods

Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

Results

Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery.

Conclusion

Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired.