慢病毒shRNA表达载体介导人肿瘤细胞基因稳定沉默的影响因素

目的 探讨慢病毒载体介导人肿瘤细胞RNA干扰的影响因素。方法 以乏氧诱导因子-1α（Hypoxia-inducible factor-1α, HIF-1α）和乏氧诱导因子-1β（Hypoxia-inducible factor-1β, HIF-1β）基因为靶基因,采用Invitrogen公司的BLOCK-iT Lentiviral RNAi Expression System生产表达靶基因shRNA的慢病毒载体,转导Hela、SPCA1和A549,采用定量RT-PCR技术检测靶基因mRNA表达水平。结果 用此系统生产慢病毒,每一10cm培养皿可收获6.3×1010个病毒颗粒。浓度为2×1010copies/ml的Lenti6-HIF1α和Lenti6-HIF1β转导SPCA1、A549和Hela细胞的功能滴度分别为：1.8×106TU/ml、1.2×106TU/ml、1.75×106TU/ml和1.76×106TU/ml、1.21×106TU/ml和1.79×106TU/ml。延长病毒的吸附时间可以提高转导效率, 8小时以内转导效率与吸附时间呈正比,12小时开始进入平台期。1/4、1/2、1、2、4、8倍MOI的Lenti6-HIF1α病毒转导SPCA1和Hela细胞48小时后,RNAi效果与病毒量呈正相比。用筛选的转导细胞证实,RNAi长期效果与细胞类型无关,但与shRNA表达结构整合到靶细胞基因组的拷贝数呈正相关。结论 慢病毒载体介导人肿瘤细胞RNA干扰,短期基因抑制效果取决于细胞类型、病毒量和病毒的吸附时间,稳定基因沉默效果与病毒整合到靶细胞基因组的拷贝数密切相关。     

HIV-1缺损慢病毒载体的高滴度制备及其介导的高效基因转移

近来,慢病毒载体引起了极大关注,己成为转基因操作中重要的工具。用编码病毒组份的三质粒系统共转染293T包装细胞系,建立了大量制备HIV-1缺损慢病毒载体的方法,病毒载体的度可达到1.1×107IU/mL,离心浓缩可将载体滴度提高100倍以上。HIV-1缺损慢病毒载体可以高效转导人淋巴瘤等多种来源的细胞,RT-PCR检测显示外源基因GFP稳定表达达18个月以上,长期传代观测未检出p24抗原蛋白或可复制病毒。     

靶向ADAM17基因RNA干扰慢病毒载体的构建及慢病毒包装

目的构建靶向ADAM17基因RNA干扰（RNAi）慢病毒载体及包装慢病毒。方法根据人ADAM17mRNA序列设计4个靶序列,合成4对寡核苷酸序列,同时合成1对阴性对照寡核苷酸序列;将以上5对寡核苷酸序列退火后连入pLVTHM质粒,经酶切和测序鉴定。将重组慢病毒质粒转染至A549细胞,以Real-time PCR检测A549细胞中ADAM17 mRNA表达。将干扰效果最佳的质粒载体和包装质粒共转染至293T细胞,包装产生病毒颗粒。以流式细胞术检测重组慢病毒的滴度。结果酶切和测序证实干扰靶序列已被准确克隆到pLVTHM质粒载体。pLVTHM-ADAM17-siRNA1-4均可显著抑制A549细胞ADAM17 mRNA的表达,其中pLVTHM-ADAM17-siRNA4的抑制效果最佳。LV-ADAM17-siRNA4重组慢病毒的滴度为2.16×108TU/ml。结论成功构建了靶向人ADAM17基因RNAi慢病毒载体及包装了重组慢病毒。     

Genomic integration occurs in the packaging cell via unexported lentiviral precursors

Objective

To use HIV-1 based lentivirus components to produce gene integration and the formation of a stable cell line in the packaging cell line without viral infection.

Results

A co-transfection of a Human Embryonic Kidney (HEK) 293 packaging cell line with Gag–pol (GP) and a transfer vector, without the envelope vector, produces a stable cell line after 2 weeks of selection. Furthermore, a matrix protein deficient GP in the packaging vector enhances this integration. This supports that, in theory, unexported lentiviral cores produced within the packaging cell can infect itself without requiring the release of any lentiviral particles.

Conclusion

If the packaging cell is also the target cell, then gene integration leading to a stable cell line can be accomplished without viral particle infection.

     

CHD5基因RNAi慢病毒载体的构建及其对结直肠癌细胞生物学功能的影响

目的:研究人类肿瘤相关基因CHD5基因miR-shRNA慢病毒对结直肠癌Lovo细胞的影响。方法:利用软件设计对CHD5基因干扰有效的序列,合成靶序列,退火形成双链DNA,酶切后与载体相连接。将重组慢病毒载体pPRIME-TET-GFP-CHD5与慢病毒包装质粒pCMV-VSV-G,pRSV-Rev,pMDLg-pRRE共转染293FT细胞,将包装重组慢病毒感染人类结直肠癌Lovo细胞。通过荧光定量PCR和Western blot验证CHD5基因在细胞中的表达情况,应用MTT检测CHD5低表达对Lovo细胞增殖的影响。结果:成功构建pPRIME-TET-GFP-CHD5重组质粒,经酶切及序列测定正确,包装的病毒滴度为3.1×106TU/ml。用制备的病毒上清感染Lovo细胞后,荧光定量PCR和Western blot分析结果显示该慢病毒可分别在转录和蛋白质水平上抑制Lovo细胞CHD5基因的表达,并使得Lovo细胞增殖失控。结论:成功构建CHD5慢病毒表达载体,表达的慢病毒可有效的感染Lovo细胞,提高Lovo细胞的增殖能力。     

Generation of a Genome Scale Lentiviral Vector Library for EF1α Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.     

TRAIL 慢病毒载体构建及其在肝癌细胞中的表达

目的:构建携带TRAIL基因的慢病毒表达载体并实现其在肝癌细胞株HepG2中的稳定高表达。方法:构建TRAIL重组慢病毒表达载体pCDH-CMV-TRAIL-EF1-GFP-T2A-Puro,脂质体法将重组慢病毒载体和包装质粒混合物共转染293T细胞,包装产生慢病毒颗粒,纯化并测定病毒滴度。利用Western blotting检测TRAIL蛋白在HepG2中的表达。结果:酶切以及测序证实,成功构建TRAIL基因重组慢病毒载体,纯化的慢病毒滴度为1.02×104ifμ/μL。利用嘌呤霉素筛选获得稳定表达TRAIL的细胞系,经Western blot方法检测到TRAIL蛋白的稳定高表达。结论:成功构建了带有TRAIL基因的慢病毒载体,并实现其在HepG2的稳定高表达。     

shRNA沉默CBS基因的慢病毒载体构建及稳定转染PC12细胞系的建立

目的:硫化氢是一种重要的气体信号分子,作为一种神经调质在神经系统中起重要作用。胱硫醚-β-合成酶(CBS)是脑内硫化氢合成的主要酶。构建针对大鼠CBS基因的shRNA干扰载体,稳定转染PC12细胞,观察该载体对PC12细胞CBS基因的沉默效应。方法:构建三条针对大鼠CBS基因的shRNA,经前期实验筛序一条最有效靶点与载体GV248(h U6-MCS-Ubiquitin-EGFP-IRES-puromycin)连接,经转化及PCR阳性克隆筛选及测序鉴定。将LV-CBS-ShRNA慢病毒载体连同包装载体经脂质体2 000共转染到293T细胞,慢病毒包装后用荧光法进行滴度测定。将包装好的慢病毒转染到PC12细胞,用嘌呤霉素进行筛选,得到稳定转染LV-CBS ShRNA的PC12细胞。实时荧光定量PCR检测CBSmRNA的表达,Western-blot检测CBS蛋白的表达。结果:PCR扩增和测序结果证明,成功构建大鼠LV-CBS ShRNA慢病毒载体,经包装产生的慢病毒滴度为1×109TU/m L。与转染阴性对照慢病毒(LV-NC-ShRNA)的细胞比较,LV-CBS ShRNA慢病毒转染可使PC12的CBSmRNA和CBS蛋白表达分别下降51.2%和48%。成功构建CBS基因ShRNA干扰的PC12细胞株,为后续研究CBS在神经系统中的作用奠定基础。     

hsa-miR-20a低表达慢病毒载体的构建及其表达鉴定

目的:构建hsa-mi R-20a低表达慢病毒载体,检测其在HL-60中表达。方法:采用In-fusion重组交换克隆法设计并合成hsa-mi R-20a前体序列的扩增引物,扩增获得目的片段插入慢病毒GV159中,得到重组的LV-hsa-mi R-20a表达载体,通过与包装质粒共转染293T细胞,获得携带hsa-mi R-20a的重组慢病毒并测定病毒滴度。取对数生长期HL-60细胞根据病毒滴度及细胞MOI值感染慢病毒,感染后24 h、48 h、72 h、96 h镜下观察荧光表达情况,判断感染效率,q RT-PCR检测HL-60细胞hsa-mi R-20a的表达变化。结果:成功构建LV-hsa-mi R-20a低表达慢病毒载体,其病毒滴度为(8E+8)TU/m L。该病毒感染HL-60细胞的效率可高达到80%,并可有效降低HL-60细胞hsa-mi R-20a表达水平。结论:成功构建了hsa-mi R-20a低表达慢病毒载体,包被的慢病毒可以在HL-60细胞中实现低表达效果,为后续功能研究奠定了基础。     

Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses

BACKGROUND: The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. METHODS: Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. RESULTS: The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. CONCLUSIONS: The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.     

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.     

Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector

Background

The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.

Methods/Principal Findings

We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.

Conclusions/Significance

This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)     

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.     

大鼠CXCR4基因RNAi慢病毒载体的构建及其在骨髓间质干细胞中的表达

为深入研究CXCR4在骨髓间质干细胞(MSCs)体内迁移中的作用, 构建CXCR4基因RNA干扰(RNAi)慢病毒载体并实现其在大鼠MSCs (rMSCs)中表达。根据大鼠CXCR4 mRNA序列, 设计并合成包含各靶序列的互补DNA链,插入pSUPER载体的H1 RNA启动子后面, 产生pRiCXCR4, 将其中的CXCR4 shRNA表达结构酶切插入慢病毒载体质粒pNL-EGFP, 产生pNL-RiCXCR4-EGFP。在脂质体介导下与包装质粒pHELPER和包膜质粒pVSVG共转染293T细胞, 包装生产慢病毒,测定慢病毒功能滴度。慢病毒转导rMSCs后, 用Real-time RT-PCR、Western blotting和流式细胞术检测RNAi组(CXCR4a、CXCR4b和CXCR4c)、空载体组(Mock)和对照组(Control)中CXCR4表达情况。结果显示, 酶切和测序证实pRiCXCR4质粒构建正确, 产生能同时表达增强型绿色荧光蛋白(EGFP)和CXCR4 shRNA的慢病毒载体质粒pNL-RiCXCR4-EGFP, 未浓缩和浓缩慢病毒悬液的功能滴度分别为6.4×104TU/mL和6.9×106TU/mL。慢病毒转导rMSCs 48 h后, 与空载体组和空白组相比, 3个RNAi组均不同程度抑制CXCR4表达, CXCR4b-MSC组在mRNA水平抑制了95.6%, 抑制作用最明显。大鼠CXCR4基因RNAi慢病毒载体构建成功, 为深入研究CXCR4在rMSCs向损伤组织定向迁移的作用奠定了基础。     

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.     

慢病毒介导的HTRA1 基因稳定过表达人脑血管平滑肌细胞株的建立

目的:构建并包装针对HTRA1基因以及其1091TC突变基因(HTRA1-Mut)的过表达慢病毒载体,以及建立稳定表达HTRA1及HTRA1-Mut基因的人脑血管平滑肌细胞(HBVSMC)株。方法:采用RT-PCR方法扩增HTRA1及HTRA1-Mut基因片段并将其连接于GV287载体质粒,采用慢病毒包装三质粒系统(GV287/p Helper 1.0/p Helper 2.0)转染293T细胞,收集富含慢病毒颗粒的细胞上清液并标定病毒滴度,慢病毒感染经培养和鉴定的HBVSMC细胞株。结果:成功构建含HTRA1及HTRA1-Mut基因的慢病毒重组载体,PCR鉴定阳性的克隆进行测序和BLAST比对分析显示与源基因序列一致,并能够有效的感染并在293T细胞中表达。表达载体包装后测定病毒滴度为:2E+8 TU/mL。过表达慢病毒感染后HBVSMC有荧光表达,并且荧光率达80%以上,细胞生长良好传后细胞几乎无死亡现象。结论:成功构建了过表达HTRA1及HTRA1-Mut基因的慢病毒表达载体,得到了较高滴度的病毒悬液,建成了稳定表达HTRA1及HTRA1-Mut基因的HBVSMC细胞株,为进一步探讨HTRA1基因及突变后细胞的功能变化提供了良好的研究工具。