目的基因慢病毒载体的构建与包装

目的探讨Oct4,Sox2,c-Myc及Klf4-慢病毒载体的构建与包装。方法从含有Oct4,Sox2,c-Myc及Klf4的质粒中获取目的基因,将目的基因与酶切线性化的慢病毒载体进行定向连接,其连接产物转化细菌感受态细胞,对长出的阳性克隆进行测序和比对分析,并对长出的阳性克隆进行PCR鉴定,通过三质粒系统共转染293T细胞对慢病毒进行包装并进行滴度测定。结果成功构建并包装出Oct4,Sox2,c-Myc及Klf4-慢病毒载体,基因测序结果与目标序列完全一致;PCR鉴定结果显示,4个目的基因均为阳性;Oct4,Sox2,c-Myc及Klf4-慢病毒载体滴度分别为1×109,1×109,1×109,2×109 TU/mL。结论通过三质粒系统可构建并包装Oct4,Sox2,c-Myc及Klf4-慢病毒载体。     

Generation of lentivirus vectors using recombinant baculoviruses

In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 x 10(6) TU ml(-1), which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.     

Characterization of HIV-1 vectors with gammaretrovirus envelope glycoproteins produced from stable packaging cells

We have recently described a novel, stable human immunodeficiency virus type 1 (HIV-1) vector packaging system, STAR. High-titre HIV-1 vectors bearing gammaretrovirus envelopes (Env) are continuously produced from STAR cells. Here we compare the properties of such vectors, with the amphotropic murine leukaemia virus (MLV-A) Env, a modified gibbon ape leukaemia virus (GALV) Env and two modified versions of the cat endogenous retrovirus RD114 Env, produced from STAR cells, to transiently produced HIV-1 vectors with vesicular stomatitis virus G protein (VSV-G). Our results indicate that gammaretrovirus pseudotypes from STAR cells are relatively stable at 37 degrees C and are resistant to inactivation by freeze/thaw cycling or incubation with human sera. HIV-1(VSV-G) was, however, sensitive to freeze/thaw when harvested in serum-free media and was readily inactivated in human sera. Furthermore, the titre of 'gamma-retrovirus' pseudotypes, but not HIV-1(VSV-G), could be increased by the use of a combination of polybrene and spinoculation. All pseudotypes could be efficiently concentrated, but soluble gammaretrovirus Env could act as an inhibitor of infection.     

HABP1基因慢病毒干扰和表达载体的构建及表达

摘要：目的：构建慢病毒介导的透明质酸结合蛋白1基因（HABP1）干扰载体及表达载体,建立HABP1降表达及过表达的稳定肾癌细胞系786-0。 方法：合成人HABP1基因短发夹RNA(shRNA)干扰序列,与慢病毒载体pLKO.1-TRC连接产生重组质粒pLKO.1-shHABP1;双酶切质粒pCDNA3.1(-)-HABP1-Flag,将目的基因片段连接到pCDH-CMV-MCS-EF1-Puro载体上,组成重组表达载体pCDH-HABP1;酶切鉴定及DNA测序后,转染293T细胞,收集慢病毒颗粒感染786-0细胞,嘌呤霉素筛选稳定细胞系,western blot验证HABP1蛋白的表达水平。 结果：构建成干扰载体pLKO.1-shHABP1及表达载体pCDH-HABP1;与随机序列对照组比较pLKO.1-shHABP1转染的786-0细胞HABP1表达水平(0.30±0.01)明显降低(t=25.98,P<0.05),pCDH-HABP1转染786-0细胞后,HABP1的表达水平（3.20±0.40）明显升高(t=10.34,P<0.05)。 结论：成功构建HABP1基因的干扰载体及表达载体,且786-0细胞可稳定降表达及过表达HABP1。     

Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.     

Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase

Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.     

Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.     

Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors

Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors.     

慢病毒载体的构建及优化

目的：针对慢病毒载体的基因组装、转导及基因表达的优化论述。及不同种属的慢病毒载体的最低活性加以说明，同时对慢病毒遗传体系的产生、转导效率和生物安全性进行总结。资料来源：应用计算机检索Pubmed以及美国ScI和Medline数据库2000-01／2004-12有关慢病毒载体的论文，检索词“lentivirus vectors，immunodeficiency”，并限定文章语言种类为English。同时应用计算机检索中国期刊网数据库1995-01／2005-12有关慢病毒载体构建的文章，限定文章语言种类为汉语，检索词“慢病毒载体”。资料选择：对检索到的相关信息及文章进行整理。选取针对性强的文章。纳入标准：①临床研究性文章。②基础研究文章。排除标准：①其他病毒载体的结构文献以及重复性资料。②诸多病毒实验应用性文献。③重复同一研究。资料提炼：共收集到198篇关于慢病毒结构原理的相关文献，其中包括英文文献150篇，中文文献48篇，有21篇符合纳入标准。资料综合：介绍不同源慢病毒载体的构建和慢病毒的基因位点。影响基因转移的一个主要因素是细胞的基因表达。人类转基因载体的一个重要特点是可以调节转基因的表达。调节转基因信号的另一个途径是运用可切割的前病毒产物。转基因载体是建立在逆转录病毒之上，主要包括肿瘤逆转录病毒和慢病毒，这些病毒载体系统是哺乳动物细胞外源基因传递、整合和表达的有效途径。结论：蛋白酶、rev．慢病毒蛋白和VSV／G糖蛋白均具有细胞毒性和抑制细胞的作用，当连续表达时要求使用诱导表达系统。     

慢病毒载体的构建及优化

目的:针对慢病毒载体的基因组装、转导及基因表达的优化论述,及不同种属的慢病毒载体的最低活性加以说明,同时对慢病毒遗传体系的产生、转导效率和生物安全性进行总结。资料来源:应用计算机检索Pubmed以及美国SCI和Medline数据库2000-01/2004-12有关慢病毒载体的论文,检索词“lentivirusvectors,immunodeficiency”,并限定文章语言种类为English。同时应用计算机检索中国期刊网数据库1995-01/2005-12有关慢病毒载体构建的文章,限定文章语言种类为汉语,检索词“慢病毒载体”。资料选择:对检索到的相关信息及文章进行整理,选取针对性强的文章。纳入标准:①临床研究性文章。②基础研究文章。排除标准:①其他病毒载体的结构文献以及重复性资料。②诸多病毒实验应用性文献。③重复同一研究。资料提炼:共收集到198篇关于慢病毒结构原理的相关文献,其中包括英文文献150篇,中文文献48篇,有21篇符合纳入标准。资料综合:介绍不同源慢病毒载体的构建和慢病毒的基因位点。影响基因转移的一个主要因素是细胞的基因表达。人类转基因载体的一个重要特点是可以调节转基因的表达。调节转基因信号的另一个途径是运用可切割的前病毒产物。转基因载体是建立在逆转录病毒之上,主要包括肿瘤逆转录病毒和慢病毒,这些病毒载体系统是哺乳动物细胞外源基因传递、整合和表达的有效途径。结论:蛋白酶、rev.慢病毒蛋白和VSV/G糖蛋白均具有细胞毒性和抑制细胞的作用,当连续表达时要求使用诱导表达系统。     

SMAD家族成员4 shRNA干扰慢病毒构建与分析

目的制备shRNA干扰人SMAD家族成员4(SMAD4)的慢病毒载体(shRNA-SMAD4),为深入研究BMPs/SMAD信号通路奠定基础。方法利用在线干扰设计网页进行干扰引物设计,根据小干扰RNA的设计原则,选取3对针对SMAD4特异性干扰引物而合成。合成的片段连接到慢病毒载体质粒pSIH1-H1,形成pSIH1-H1-shRNA-SMAD4质粒。用PCR及测序对其鉴定,然后与慢病毒包装质粒混匀,由脂质体转染至HEK293T细胞进行包装,产生慢病毒shRNA-SMAD4,并收集上清液感染乳腺癌MDA-MB-231细胞,通过RT-PCR及Western Blot鉴定重组慢病毒干扰效果。结果(1)shRNA干扰SMAD4特异性引物的获得。(2)pSIH1-H1-shRNA SMAD4质粒经PCR验证和测序后,证实构建成功。(3)shRNA-SMAD4在HEK293T中重组成慢病毒,并成功感染MDA-MB-231。(4)shRNA-SMAD4感染乳腺癌MDA-MB-231细胞后SMAD4的表达降低。结论成功构建shRNA-SMAD4慢病毒载体,并能有效抑制SMAD4表达。     

慢病毒高效感染皮肤人永生化表皮细胞株HaCaT细胞的分化状态

背景：研究证实YY1主要在小鼠基底层未分化表皮细胞中表达,且随着表皮细胞向基底上层分化其表达逐渐下降,这种差异性表达方式说明YY1可能是表皮细胞分化过程中重要的调节因子之一。目的：采用慢病毒-YY1感染HaCaT细胞观察YY1过表达对细胞分化的影响。方法：将慢病毒-YY1感染至HaCaT细胞,经puromycin筛选,建立单克隆稳定细胞株,设对照组为慢病毒感染的HaCaT细胞和未感染的HaCaT细胞,Western blot检测分析YY1蛋白的表达水平;将慢病毒-YY1-HaCaT组和HaCaT-YY1组细胞各分成2组,一组在低钙（0.12 mmol/L）培养基条件下培养48 h,另一组在低钙（0.12 mmol/L）培养基条件下培养24 h后在高钙（0.35 mmol/L）培养基条件下再培养24 h,用Western blot法检测YY1过表达对HaCaT细胞分化中的表皮细胞特异性分化角蛋白K1、K10、K14和晚期分化产物外皮蛋白、中间丝相关蛋白、兜甲蛋白的影响。结果与结论：慢病毒-YY1成功感染HaCaT细胞,高钙条件下获得的单克隆细胞株过表达YY1蛋白,可抑制K1、外皮蛋白和兜甲蛋白的合成,从而阻止表皮细胞角质化进程并使细胞处于未分化状态。结果说明慢病毒可高效感染皮肤人永生化表皮细胞株HaCaT细胞,YY1可能是抑制基底表皮细胞分化并维持其未分化增殖状态的重要因子之一。     

慢病毒载体有效转移并感染心肌成纤维细胞

背景:心肌成纤维细胞过度增殖、胶原合成增多是心肌纤维化的主要病理基础.慢病毒载体系统是一种新型的基因治疗转移系统,能够高效感染分裂期和非分裂期细胞.目的:建立慢病毒载体有效转移并表达于大鼠心肌成纤维细胞的方法,为利用慢病毒载体介导心肌成纤维细胞的基因靶向治疗奠定基础.设计、时间及地点:对比观察,实验予2008-08/10在华中科技大学同济医学院附属协和医院老年病及上海吉凯公司实验室完成.材料:新生1～3日的SD大鼠乳鼠30只;Lentivirus载体、Enhanced Infection Solution(ENi.S)、Polybrene均为上海占凯基因化学技术有限公司产品.方法:采用差速贴壁法培养乳鼠心肌成纤维细胞,在DMEM加入3个梯度Lentivirus感染细胞,将病毒复感染指数为200,20,2的3组细胞,每组分别加入①Lentivirus.②在细胞感染时添加polybrene提高感染效率.⑨在上述基础上加入具有促进病毒感染的细胞增强液(Enhanced Infection Solution(ENi.S)),感染4 d.主要观察指标:①心肌成纤维细胞形态特征.②倒置显微镜观察绿色荧光蛋白表达情况及感染效率.结果:成功建立心肌成纤维细胞培养模型,当慢病毒对心肌成纤维细胞的复感染指数为20时,可以达到80%的感染效率,然而必须在培养基内加入多聚阳离子转染试剂Polybrene,以提高慢病毒载体的感染效率.结论:慢病毒载体较易感染心肌成纤维细胞,是具有发展潜力的心肌成纤维细胞基因靶向治疗的新载体.     

携带Bcl-xl基因的重组慢病毒的制备和鉴定

目的制备携带Bcl-xl基因的重组慢病毒,并鉴定其感染细胞有效性。方法从本所以前构建的重组质粒pAAV-Bcl-xl获得Bcl-xl基因的编码序列,将其置换掉慢病毒质粒pSin-EF2-SOX2-Pur中的SOX2基因编码序列,从而得到重组慢病毒质粒pSin-EF2-Bcl-xl-Pur。然后利用这一质粒包装制备了携带Bcl-xl基因的重组慢病毒颗粒,用其感染细胞后用Western blot和流式检测仪分析了感染前后细胞中Bcl-xl基因的表达情况。结果感染后细胞中Bcl-xl蛋白的表达水平显著高于感染前。结论携带Bcl-xl基因的重组慢病毒颗粒包装成功,能高效感染细胞,为后续的遗传改造树突细胞打下了基础。     

Real-time fluorescence tracking of dynamic transgene variegation in stem cells.

Transgene variegation is caused by epigenetic switching between expressing and silent states. gamma-retrovirus vectors can be variegated in stem cells, but the dynamics of epigenetic remodeling during transgene variegation are unknown. Here, we measured variegated enhanced green fluorescent protein gamma-retrovirus expression over 4 days in individual embryonic stem cells while tracking cells in order to create expression lineage trees: 56 colony founder cells and their progeny were tracked over seven generations. Nineteen lineages produced synchronized inheritable trajectories of transgene silencing or reactivation, indicative of epigenetic remodeling with long-term stable inheritance. Short-term fluctuations in fluorescence intensity were also observed, which contributed low-amplitude variation to transgene expression level. These two processes have different frequencies and inheritability, but together contribute to variegated transgene expression. Inhibition of DNA methylation with 5-azacytidine eliminated long-term transgene silencing over 4 days, but short-term fluctuations continued. Our approach applies real-time imaging technology to track the long-term dynamics of transgene expression to investigate the timing and expression patterns leading to variegation.     

慢病毒载体感染人皮肤成纤维细胞的研究

目的:建立慢病毒-绿色荧光蛋白载体有效感染人包皮成纤维细胞的方法,为利用慢病毒载体介导外源基因导入人包皮成纤维细胞,后者直接诱导分化为多能干细胞的研究奠定基础.方法:采用0.1%Ⅰ型胶原酶冷消化联合组织块贴壁法培养人包皮成纤维细胞,在细胞培养液中分别加入不同感染复数的慢病毒和具有促进病毒感染的细胞增强液,并于感染48 h后观察慢病毒转导情况.结果:成功建立人包皮成纤维细胞培养模型,当慢病毒对人包皮成纤维细胞的感染复数为20及在培养基中加入细胞增强液时,感染效率可以达到80%,能满足后续实验的需要.结论:慢病毒较易感染人包皮成纤维细胞,是具有发展潜力的多能干细胞细胞技术研究的新载体.