NT-3基因过表达慢病毒载体的构建和包装及鉴定

目的构建神经营养因子-3(neurotrophin-3,NT-3)基因慢病毒载体,检测其在大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)中的表达。方法体外扩增NT-3,将NT-3全长载体GV287-GFP与扩增出的NT-3用AgeI进行酶切,将NT-3全长序列克隆入GV-287-GFP,转化大肠杆菌DHS a感受态细胞,筛选出阳性克隆进行基因测序。重组GV287-EGFP质粒、pHelper 1.0质粒和pHelper 2.0质粒三质粒共转染至包装细胞293T,培养48 h后收集细胞上清液,将病毒浓缩后在293T细胞中测定病毒滴度,并检测慢病毒载体在MSCs的转染效率。荧光显微镜观察转染是否成功,RT-PCR和Western blot检测MSCs细胞中NT-3蛋白的表达。结果测序结果和Western blot检测均证明NT-3慢病毒载体构建正确,且在细胞中正确表达。与辅助质粒共包装细胞获得慢病毒颗粒,并成功感染MSCs细胞。包装慢病毒、浓缩病毒悬液的滴度为2×109/ml慢病毒浓缩液。结论成功构建了稳定高效表达NT-3基因的慢病毒载体。     

大鼠硫酸软骨素蛋白多糖基因shRNA慢病毒载体的构建及干扰效率鉴定*☆

背景：最近研究发现硫酸软骨素蛋白多糖(NG2)在中枢神经系统中参与多种生理病理功能,慢病毒载体可感染分裂期细胞或非分裂期的细胞,并能在细胞内高效稳定的表达。 目的：构建大鼠源性NG2基因shRNA慢病毒载体并检测其干扰效率。 方法：选择大鼠NG2基因RNA干扰的靶序列,合成Oligo DNA,退火形成双链DNA,与Hpa Ⅰ和Xho Ⅰ双酶切后的pFU-GW-RNAi载体连接产生pLV-NG2-RNAi,PCR筛选阳性克隆,测序鉴定。将重组载体与pHelper 1.0载体、pHelper 2.0载体通过lipofectamineTM 2000共转染293T细胞包装产生慢病毒LV-NG2-RNAi,收集病毒上清并浓缩。采用孔稀释滴度测定法计算病毒滴度。将LV-NG2-RNAi慢病毒感染C6细胞,于感染后96 h提取细胞总蛋白,采用Western blot检测NG2的表达。 结果与结论：经PCR和测序证实构建片段大小及DNA序列与目的序列一致,实验成功构建大鼠NG2基因shRNA慢病毒载体LV-NG2-RNAi。包装浓缩慢病毒的滴度为8×1011 TU/L。Western blot检测显示在感染复数为50时,感染LV-NG2-RNAi慢病毒的C6细胞较感染对照慢病毒及未感染细胞NG2的表达明显降低,干扰效率可达100%(P < 0.05)。结果证实了实验成功构建大鼠NG2基因shRNA慢病毒载体,且该载体能够在细胞水平有效沉默靶基因。     

沉默Toll样受体4基因表达的RNAi慢病毒载体的构建******

背景：Toll样受体4 (toll-like receptor4,TLR4)是介导内毒素/脂多糖应答的主要受体,在由内毒素诱导的炎性反应的信号通路中发挥着重要作用。 目的：构建人TLR4基因的RNA干扰慢病毒载体,并观察其对人脐静脉内皮细胞TLR4在蛋白水平的沉默效应。 方法：利用Invitrogen在线软件设计人TLR4基因shRNA序列,合成、退火形成双链寡核苷酸后克隆到线性载体pENTRTM/H1/TO的黏性末端,并进行DNA测序。得到的阳性重组子再与慢病毒载体进行重组反应,从而获得干扰TLR4基因真核表达的慢病毒载体。在脂质体的介导下将慢病毒包装辅助复合体和TLR4基因的真核表达慢病毒载体导入293FT细胞包装病毒,测定病毒滴度,感染人脐静脉内皮细胞,检验其干扰TLR4基因表达的有效性。 结果与结论：实验成功构建TLR4基因真核表达慢病毒干扰载体并获得相应的慢病毒,病毒滴度为8.7×106 U/mL。免疫印迹杂交结果表明,所获得的慢病毒感染人脐静脉内皮细胞TLR4基因在蛋白水平的表达显著降低。实验成功构建了人TLR4基因慢病毒RNA干扰表达载体,并验证了其在人脐静脉内皮细胞上的有效性。     

Nesprin基因RNAi慢病毒载体的构建*

背景：Nesprin蛋白缺失将影响细胞骨架组织和动态平衡,引起细胞骨架刚性丧失或导致细胞过早成熟老化,其对间充质干细胞的作用如何？ 目的：构建Nesprin蛋白siRNA慢病毒载体,并转染骨髓间充质干细胞。 方法：针对Nesprin靶基因序列设计并合成4对miRNA oligo,将4种miRNA干扰质粒转入大鼠血管平滑肌细胞,筛选最有效干扰序列;将最佳干扰序列和pDONR221载体进行重组反应,获得含干扰序列的入门载体,再将入门载体和慢病毒表达目的载体pLenti6/V5-DEST进行重组反应,获得含干扰序列的慢病毒表达载体,转染包装细胞293T细胞,包装慢病毒,以293T细胞GFP蛋白水平测定病毒滴度。慢病毒转染大鼠骨髓间充质干细胞。 结果与结论：测序证实合成的4对miRNA oligo正确,RT-PCR和western-blot筛选出最佳干扰miRNA质粒为SR-3,成功构建了Nesprin siRNA的慢病毒载体LV-siNesprin。包装慢病毒,浓缩病毒悬液的活性滴度为106 TU/mL。慢病毒成功了转染骨髓间充质干细胞细胞。     

人EGFL7基因短发夹结构RNA慢病毒载体的构建及鉴定

目的构建人类表皮生长因子域7（EGFL7）基因RNA干扰（RNAi）慢病毒载体。方法将靶向EGFL7基因的短发夹结构RNA（shRNA）表达序列连接到包含U6启动子及绿色荧光蛋白（GFP）报告基因的慢病毒载体pGCL-GFP中,获得重组质粒,命名为pGCL-GFP-vshEGFL7,经多聚酶链反应（PCR）和测序鉴定后,与慢病毒包装质粒pHelper 1.0及pHelper 2.0通过lipofectamine 2000共转染至包装细胞293T,包装产生病毒液,测定其滴度。结果PCR扩增和测序结果证实EGFL7shRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液的滴度为4.8×107TU/ml。结论成功构建人EGFL7基因shRNA慢病毒载体。     

表达IL -1ra的慢病毒载体的构建与鉴定

目的 构建表达IL - 1ra的慢病毒载体,为神经前体细胞(NPCs)的基因修饰移植治疗脊髓损伤的研究提供基础.方法 从IL - 1ra质粒中克隆出IL -1ra基因,下游融合IRES2 - EGFP,装载于pLenti6.3 V5 - DEST中,获得含目的序列IL1ra - IRES2 - EGFP的慢病毒质粒,测序并包装;病毒感染HEK293细胞,通过GFP表达水平测定病毒滴度.并转染NPCs,观察NPCs的生长情况.结果 测序证实,用引物CMV -F和IRES2 -R扩增的质粒中IL -1ra的DNA序列与Genebank中序列完全一致;慢病毒悬液的滴度为3.5×106 TU/ml;NPCs转染重组慢病毒后且未出现生长特性的改变.结论 成功构建了表达IL - 1ra的慢病毒载体pLenti6.3- IL1ra - IRES2 - EGFP.     

应用Cyclin D1和bcl-2为靶标的siRNA慢病毒载体构建和鉴定

背景：近年来,与骨肉瘤耐药相关的基因研究大多局限于单个基因或单个通路。而进行细胞凋亡和细胞周期调控双通道同时阻断有可能逆转药物耐受机制。 目的：构建bcl-2和cyclin D1特异性siRNA慢病毒载体,拟将其转入骨肉瘤耐药细胞株,探讨对骨肉瘤耐药性的逆转作用。 方法：采用限制性内切酶酶切、T4DNA连接酶连接等方法,将bcl-2和cyclin D1基因分别插入慢病毒载体pSIH1-H1-copGFP shRNA Vector中,构建bcl-2和cyclin D1与pSIH1-H1-copGFP共表达的慢病毒载体(pSIH1-H1-copGFP- bcl-2-siRNA和pSIH1-H1-copGFP-cyclinD1-siRNA)。构建成功后的慢病毒质粒系统和pPACK包装质粒共转染293T细胞,过滤,浓缩病毒,利用荧光蛋白作为报告基因,对病毒滴度和感染效率进行检测。 结果与结论：4对bcl-2和cyclin D1特异性siRNA与双酶切慢病毒载体pSIH1- H1-copGFP shRNA Vector连接成功。共转染293T细胞包装病毒并浓缩后滴度达1.14×104 ifu/μL,适合感染目的细胞。实时荧光定量PCR检测结果显示对bcl-2和cyclinD1基因的干扰效率最高分别达88%和87%。证实将siRNA技术应用于bcl-2和cyclin D1,能够构建出有效的bcl-2和cyclin D1特异性siRNA慢病毒载体。     

小鼠TNF-α基因RNAi慢病毒载体的构建及体外研究

目的为探讨TNF-α在C型Niemann-Pick病（NPC）神经变性中的作用,构建携带小鼠TNF-α-siRNA的慢病毒载体并进行体外研究。方法设计3个TNF-α-siRNA序列和1个无关对照序列,克隆到酶切的pSIH1-H1-copGFP siRNA载体;重组质粒转染293T细胞,通过实时定量PCR检测筛选出干扰效果最佳的TNF-α-siRNA;筛选出的pSIH-siRNA、pSIH-negative分别与慢病毒包装质粒共转染293T细胞生成慢病毒;慢病毒感染BV-2细胞和星形胶质细胞,采用RT-PCR以及Elisa检测TNF-α-siRNA的沉默效果。结果成功构建携带TNF-α-siRNA的慢病毒载体,滴度达2×10^8ifu／L;慢病毒感染BV-2和星形胶质细胞,表达GFP且可下调TNF-α表达。结论TNF-α-siRNA能明显下调TNF-α的表达,为研究和治疗神经变性疾病以及TNF-α相关疾病提供了有力的工具。     

新型慢病毒载体的构建及其在人骨髓间充质干细胞基因转导中的应用

背景：构建一个组成性表达某特定基因同时携带荧光报告基因和抗生素筛选基因的慢病毒载体目前未见报道。 目的：观察新型慢病毒载体pLVpuro/EF1α-PEDF-IRES-EGFP在人骨髓间充质干细胞基因转导中的表达。 方法：通过PCR在PEDF基因的两端加上attB位点,构建表达载体pLVpuro/EF1α-PEDF-IRES-EGFP,将表达载体与包装质粒(ViraPowerTM Lentiviral Packaging Mix)共转染293FT细胞。通过多次感染的方法将慢病毒载体导入人骨髓间充质干细胞,转导后第7天开始使用1~5 mg/L嘌呤霉素筛选5 d,得到表达PEDF和EGFP的人骨髓间充质干细胞,并进行Western、Elisa的鉴定分析。 结果与结论：经PCR和测序证实,慢病毒表达载体pLVpuro/EF1α-PEDF-IRES-EGFP构建成功;将其成功导入人骨髓间充质干细胞,经过筛选获得纯化的过表达PEDF基因的绿色荧光细胞群。     

hTfR基因在体外神经干细胞的表达研究

目的将人转铁蛋白受体(hTfR)基因克隆到慢病毒表达载体pLENTI6.3,鉴定其正确性,体外感染小鼠神经干细胞并检测其表达,为活体神经干细胞(NSCs)MR分子成像提供实验基础。方法利用聚合酶链反应技术(PCR)扩增hTfR基因,并克隆到pLENTI6.3载体;通过菌落PCR初步筛选、BamH1酶切鉴定和测序鉴定构建的pLENTI6.3-hTfR-IRESEGFP重组载体,利用Lipofectin2000试剂将PLP1、PLP2、PLP-VSVG和pLenti6.3-hTfR-IRES-EGFP共转染293T细胞进行慢病毒包装,48h后收集病毒上清。体外感染NSCs,Western bolt检测hTfR的表达。结果成功构建了hTfR基因慢病毒表达载体,包装的慢病毒颗粒成功感染NSCs,Western bolt鉴定hTfR在NSCs过表达。结论 pLENTI6.3-hTfR-IRES-EGFP慢病毒表达载体构建成功,并建立其慢病毒表达系统,为下一步进行活体干细胞MR分子成像实验研究奠定基础。     

Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins

For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease.     

Helper virus-free HSV-1 vectors packaged both in the presence of VSV G protein and in the absence of HSV-1 glycoprotein B support gene transfer into neurons in the rat striatum

Herpes simplex virus (HSV-1) vectors have potential for gene transfer into quiescent cells, but the gene transfer process could be more efficient. In other vector systems, both the titers and the efficiency of gene transfer have been enhanced by pseudotyping the vector particles with vesicular stomatitis virus (VSV) G protein. In this report, we pseudotyped helper virus-free HSV-1 plasmid vectors with VSV G protein. Packaging was performed in the presence of both VSV G protein and a deletion in an essential HSV-1 glycoprotein, gB. The resulting vector stocks supported gene transfer into both fibroblast and neuronal cell lines. VSV G protein was required for gene transfer because preincubation of these vector stocks with antibodies directed against either VSV G protein or VSV reduced the titer to undetectable levels. Although the titers were lower than those obtained using the unmodified vector system, the titers were not increased by use of chimeric proteins that contain the extracellular domain of VSV G protein and the transmembrane and/or cytoplasmic domains of specific HSV-1 glycoproteins. Also, the titers were not increased by performing the packaging in the presence of deletions in multiple HSV-1 glycoproteins. Nonetheless, pHSVlac pseudotyped with VSV G protein supported gene transfer into striatal neurons in the rat brain. Thus, HSV-1 vectors pseudotyped with VSV G protein may be useful for specific gene transfer studies.     

High Transgene Expression by Lentiviral Vectors Causes Maldevelopment of Purkinje Cells In Vivo

Lentiviral vectors are promising as gene-transfer vehicles for gene therapy targeted to intractable brain diseases. Although lentiviral vectors are thought to exert little toxicity on infected cells, the adverse influence of viral infection on vulnerable developing neurons has not been well studied. Here, we examined whether lentiviral vector infection and subsequent transgene expression affected the morphological and functional maturation of vigorously developing cerebellar Purkinje cells in vivo. Lentiviral vectors expressing GFP under the control of the murine stem cell virus (MSCV) promoter were injected into the cerebellar cortex of neonatal rat pups. Three weeks after treatment, GFP-expressing Purkinje cells were compared with control Purkinje cells from phosphate-buffered saline-injected rats. Analysis of the dendritic tree showed that total dendrite length in GFP-expressing Purkinje cells was almost 80% that in control Purkinje cells. Electrophysiological examination showed that short-term synaptic plasticity at parallel fiber–Purkinje cell synapses and climbing fiber–Purkinje cell synapses was significantly altered in GFP-expressing Purkinje cells. In contrast, maldevelopment of infected Purkinje cells was substantially attenuated when lentiviral vectors with much weaker promoter activity were used. These results suggest that the maldevelopment of Purkinje cells was mainly caused by subsequent expression of a high amount of GFP driven by the strong MSCV promoter. Thus, the use of lentiviral vectors carrying a strong promoter may require particular precautions when applying them to neurological disorders of infants.     

Lentiviral vectors for gene delivery to normal and demyelinated white matter

Lentiviral vectors are increasingly used for gene delivery to neurons and in experimental models of neurodegeneration. Their use in gene delivery to white matter and their potential value in preventing or repairing CNS demyelination has received less attention. Here we show using a VSV-G-pseudotyped HIV-derived vector expressing the marker gene LacZ that lentiviral vectors transduce the major macroglial cell types present in normal white matter (astrocytes, oligodendrocytes, and oligodendrocyte progenitors). Injection of lentiviral vectors causes an inflammatory response at the injection site characterized by OX42(+) and ED1(+) macrophages, but only a few CD8(+) and no CD4(+) lymphocytes, and mild demyelination. Injection of lentiviral vectors into areas of toxin-induced demyelination resulted in significant numbers of cells expressing the marker gene and was a more effective means of gene delivery than was a LacZ-expressing murine retroviral vector.     

Lentiviral vector-based models of amyloid pathology: from cells to animals

Lentiviral vectors are efficient tools for the introduction of genes into a wide range of established and primary cells in vitro, ex vivo, and in vivo, and also permit efficient transgenesis in a wide range of mammalian species. Our goals have been to apply the broad capabilities of the lentiviral vector system to AD research. Using a set of vectors expressing APP and PS1 genes, we demonstrated the efficiency and fidelity of the system for in vitro biochemical analyses of genes and pathways involved in plaque deposition. These analyses were performed in cell lines and in primary neuronal cultures, which have previously been difficult to use. The methods and tools described here are applicable to the study of effects of other genes and gene combinations on APP processing, including suppression of gene activity by delivering shRNAs. We have attempted to create local plaque pathology by stereotactic injection of APP and PS1 expressing vectors into mouse brains for use as a rapid model for plaque pathology that can be used in a broad range of mammals. No amyloid or preamyloid pathology has been detected over a six-month period; the possible reasons are discussed. Lastly, we have used the vectors to create transgenic rats expressing mutant APP and mutant PS1 and have obtained the first set of positive pups with more expected. The results presented here demonstrate the utility of Lentiviral vector-based approaches to the study of AD and other neurodegenerative diseases.     

Herpes vector-mediated expression of proenkephalin reduces bone cancer pain

We examined whether a herpes simplex virus vector that expresses human proenkephalin could be used to attenuate nociception in a model of bone cancer pain in mice. Osteolytic sarcoma cells were implanted into the medullary space of the right femur, followed by a subcutaneous inoculation of a replication-defective herpes simplex virus vector expressing human proenkephalin (vector SHPE) or a lacZ-expressing control vector (vector SHZ). SHPE-inoculated mice demonstrated a significant, naltrexone-reversible decrease in pain-related behavior assessed during open-field motor activity. These results suggest that gene transfer with an enkephalin-expressing vector may be used to treat pain resulting from cancer in bone.     

Nurr1基因在体外培养大鼠骨髓源性神经干细胞的表达

目的获得表达孤儿核受体（Nurr1）基因的骨髓源性神经干细胞（BMSCs-NSCs）。方法构建携带Nurr1基因的重组腺相关病毒（AAV）载体AAV-pcDNA3．1-Nurr1，提取质粒，用脂质体转染法转染大鼠BMSCs-NSCs，并用RT-PCR和免疫细胞化学方法检测阳性细胞。结果经酶切鉴定和DNA测序，证实得到了序列正确的重组pAAV-Nurr1，获得了Nurr1阳性的BMSCs-NSCs。结论重组AAV携带的Nurr1基因能够在BMSCs-NSCs中表达，本研究为进一步探讨将该基因工程细胞用于帕金森病基因治疗提供了可能性，     

重组人生长激素基因细胞移植

目的 通过重组人生长激素基因细胞移植为生长激素缺乏症（GHD）基因治疗的生物学表达研究奠定基础。方法 构建pLXSNhGH人生长激素（hGH)逆转录病毒表达载体后,脂质体转染包装细胞系PA317,提取包装细胞上清的病毒后,将病毒感染原代小鼠胚胎成纤维细胞,将细胞移植于小鼠腹腔内,观察hGH的表达情况。结果 hGH体内持续表达达2个月以上,但由于高滴度抗hGH抗体的影响,表达水平呈波动性。结论 重组人生长激素基因原代成纤维细胞移植体内的持续表达,为细胞移植系统引入GHD基因治疗的研究奠定了实验基础。