表达红色荧光蛋白的小鼠淋巴瘤EIA细胞株的建立及鉴定

本研究通过慢病毒载体将红色荧光蛋白（DsRed）导入小鼠淋巴瘤EL4细胞,建立稳定高表达DsRed的EL4／DsRed细胞株,为建立携带红色荧光标记的小鼠肿瘤模型奠定基础。构建含新霉素耐药基因（neo）、内部核糖体进入位点序列（IRES）及DsRed的双顺反子自身失活型慢病毒载体。采用脂质体转染法将慢病毒三质粒包装系统共转染人胚肾细胞系293FT包装细胞,收集病毒上清,感染EL4细胞;利用G418的药物选择特性筛选感染细胞获得稳定表达DsRed的EL4细胞株,并扩大培养。结果表明：成功构建慢病毒表达质粒pXZ208-neo-IRES-DsRed,包装的重组慢病毒滴度可迭10^6U／ml。病毒感染EL4细胞,经终浓度为600μg／ml的G418成功筛选出稳定携带DsRed的EL4／DsRed细胞株;荧光显微镜观察及流式细胞仪检测证实该细胞株长期稳定高表达DsRed。结论：通过表达DsRed的慢病毒载体感染ELA细胞,获得了稳定高表达DsRed的EL4／DsRed细胞株。     

小鼠CXCR4基因慢病毒载体构建与真核表达

本研究旨在克隆小鼠CXC型趋化因子受体4（CXCR4）基因,构建携带增强型绿色荧光蛋白（EGFP）的CXCR4重组慢病毒载体并在真核细胞中表达。采用逆转录-聚合酶链式反应（RT-PCR）以C57BL／6小鼠骨髓细胞为模板克隆全长型CXCR4基因,连接至克隆载体pCR-Blunt,经限制性内切酶酶切后亚克隆至慢病毒转移载体,构建携带EGFP及CXCR4双顺反子结构的自身失活型重组慢病毒表达质粒;同时构建LV-IRES-EGFP作为对照质粒;通过脂质体转染法与包装质粒及包膜蛋白质粒共转染包装细胞293FT,超速离心浓缩病毒颗粒后转染293FT细胞,采用荧光显微镜和流式细胞术（FCM）检测EGFP的表达,Western blot鉴定CXCR4蛋白表达。结果表明,成功克隆小鼠CXCR4基因,构建重组慢病毒转移质粒LV-IRES-EGFP-CXCR4及对照质粒LV-IRES-EGFP,三质粒系统共转染293FT细胞后获得病毒滴度可达到10^8TU／ml。以重组慢病毒颗粒感染293Fr细胞后第3天,在荧光显微镜下观察到较强绿色荧光表达,FCM检测显示感染效率可达95％,FCM及Western blot结果显示感染后293Fr细胞表达CXCR4蛋白。结论：成功构建自身失活型慢病毒载体LV-IRES-EGFP-CXCR4,并在真核细胞中获得表达。     

Tiam1基因慢病毒表达载体构建及其在HT29细胞中的表达

目的:构建Tiam1与绿色荧光蛋白(GFP)融合基因慢病毒表达载体,观察其在人结直肠癌HT29细胞中的表达.方法:通过酶切Tiam1/C1199HA质粒获得Tiam1 cDNA片段,并将其克隆到慢病毒载体表达质粒pCDF1-copGFP中,经酶切、测序鉴定.慢病毒表达质粒和包装质粒共转染293FT细胞,获得携带Tiam1和GFP融合基因的重组慢病毒.取病毒上清感染人结直肠HT29细胞株,荧光显微镜检测绿色荧光蛋白及免疫细胞化学检测Tiam1在靶细胞中的表达.结果:重组慢病毒质粒pCDF1-Tiam1-copGFP酶切鉴定结果与目的基因条带吻合,克隆测序结果与NCBI收录的Tiam1基因序列(NM_003253)完全一致.重组慢病毒质粒可高效转染293FT细胞,病毒包装效率为88.34%.收获慢病毒上清可高效感染HT29细胞,荧光显微镜下可观察到大量绿色荧光,感染效率为55.89%,感染后靶细胞中Tiam1稳定高表达.结论:成功构建了携带Tiam1与GFP融合基因的慢病毒表达栽体pCDF1-Tiam1-copGFP,并使目的基因在靶细胞中得到稳定表达,为进一步研究Tiam1基因的相关功能提供了优质的稳定转染载体.     

稳定表达bcr-abl融合基因片段的鼠SP2/0细胞系的建立

为了建立稳定表达bcr-abl融合基因片段的SP2/0细胞系,从重组克隆载体pGEMbcr-abl中酶切出bcr-abl融合基因片段,并将其亚克隆进逆转录病毒载体pLXSN中。脂质体介导重组逆转录病毒载体pLXSNbcr-abl转染包装细胞PT67,采用G418筛选后获得稳定产病毒的包装细胞。收集病毒感染NIH/3T3细胞,加G418筛选后进行逆转录病毒滴度的测定,计算病毒效价为2×107CFU/ml。结果表明,收集病毒上清感染SP2/0细胞(H-2d),经G418筛选获得了稳定表达bcr-abl融合基因片段的SP2/0细胞株。经特异性PCR扩增和RT-PCR反应扩增,从基因组整合和基因表达水平证实获得了能稳定表达bcr-abl融合基因片段的鼠SP2/0细胞系。结论:成功建立了表达bcr-abl融合基因的鼠SP2/0细胞系,这一肿瘤细胞模型可作为研究bcr-abl基因疫苗的有效实验工具,为检验bcr-abl基因疫苗激发小鼠CTL应答的研究奠定物质基础。     

人miR-152基因的慢病毒载体的构建及稳定株筛选

目的构建人miR-152基因重组慢病毒载体并筛选稳定表达株。方法 PCR扩增包括miR-152前体所在区域的基因组DNA,克隆到慢病毒载体表达质粒pGIPZ中,得到重组的pGIPZ-miR-152表达载体,通过与包装质粒共转染HEK-293T细胞,获得携带miR-152 minigene的重组慢病毒。用病毒感染HepG2细胞,72 h后用嘌呤霉素筛选。用real-time PCR法检测miR-152基因的表达。结果构建的重组质粒经双酶切验证和测序比对鉴定正确;该质粒转染293T细胞获取的慢病毒滴度达7.5×107TU/mL;用病毒感染HepG2细胞,感染效率达70%以上。药物筛选10 d后,获得的过表达株miR-152表达量比正常细胞高近10倍。结论成功构建人miR-152基因慢病毒载体质粒pGIPZ-miR-152,并筛选出miR-152过表达细胞株,为后续实验奠定基础。     

趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达

背景：趋化因子受体7（chemokine receptor-7,CCR7）是树突状细胞从外周迁移至淋巴系统发挥作用的最重要的启动和调节者,但未成熟树突状细胞表面不表达CCR7,因此利用携带CCR7基因的未成熟树突状细胞可以更好地诱导免疫耐受。目的：构建携带小鼠CCR7基因的绿色荧光蛋白重组慢病毒载体,观察其在未成熟树突状细胞中的表达。方法：采用RT-PCR扩增小鼠CCR7基因并克隆至pCR-Blunt载体。将CCR7DNA片段及IRES-GFP连入慢病毒转移质粒LV-Lac,生成重组慢病毒质粒LV-CCR7。采用脂质体转染法将慢病毒系统3质粒（重组慢病毒质粒LV-CCR7、包装质粒ΔNRF及包膜质粒pVSVG）共转染包装慢病毒,重组慢病毒感染未成熟树突状细胞,光学显微镜观察细胞状态,流式细胞术鉴定CCR7蛋白的表达。结果与结论：实验成功扩增出小鼠CCR7DNA片段并克隆至pCR-Blunt载体,亚克隆构建慢病毒表达载体LV-CCR7,经3质粒包装系统感染293FT细胞后,24h于荧光显微镜下均观察到绿色荧光蛋白阳性表达,病毒滴度为108U/L以上,获得携带CCR7基因的重组慢病毒。慢病毒颗粒可有效感染未成熟树突状细胞,荧光显微镜可见大量GFP蛋白表达,阳性细胞达50%,流式细胞术检测到CCR7蛋白表达,LV-CCR7基因修饰的未成熟树突状细胞仍保持在未成熟状态。结果证实,实验成功构建携带小鼠CCR7基因绿色荧光慢病毒载体LV-CCR7,并可在未成熟树突状细胞细胞中表达。     

趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达

背景:趋化因子受体7(chemokine receptor-7,CCR7)是树突状细胞从外周迁移至淋巴系统发挥作用的最重要的启动和调节者,但未成熟树突状细胞表面不表达CCR7,因此利用携带CCR7基因的未成熟树突状细胞可以更好地诱导免疫耐受。目的:构建携带小鼠CCR7基因的绿色荧光蛋白重组慢病毒载体,观察其在未成熟树突状细胞中的表达。方法:采用RT-PCR扩增小鼠CCR7基因并克隆至pCR-Blunt载体。将CCR7DNA片段及IRES-GFP连入慢病毒转移质粒LV-Lac,生成重组慢病毒质粒LV-CCR7。采用脂质体转染法将慢病毒系统3质粒(重组慢病毒质粒LV-CCR7、包装质粒ΔNRF及包膜质粒pVSVG)共转染包装慢病毒,重组慢病毒感染未成熟树突状细胞,光学显微镜观察细胞状态,流式细胞术鉴定CCR7蛋白的表达。结果与结论:实验成功扩增出小鼠CCR7DNA片段并克隆至pCR-Blunt载体,亚克隆构建慢病毒表达载体LV-CCR7,经3质粒包装系统感染293FT细胞后,24h于荧光显微镜下均观察到绿色荧光蛋白阳性表达,病毒滴度为108U/L以上,获得携带CCR7基因的重组慢病毒。慢病毒颗粒可有效感染未成熟树突状细胞,荧光显微镜可见大量GFP蛋白表达,阳性细胞达50%,流式细胞术检测到CCR7蛋白表达,LV-CCR7基因修饰的未成熟树突状细胞仍保持在未成熟状态。结果证实,实验成功构建携带小鼠CCR7基因绿色荧光慢病毒载体LV-CCR7,并可在未成熟树突状细胞细胞中表达。     

PTEN基因重组慢病毒的构建

目的:构建PTEN基因重组慢病毒,转染真核细胞Ishikawa并检测PTEN基因在真核细胞内的表达.方法:分子克隆重组人PTEN基因与含有CMV启动子和绿色荧光蛋白(GFP)的慢病毒载体PLIG,PCR筛选阳性克隆,测序鉴定.将重组子PLIG-PTEN和包装质粒共转染包装细胞293T细胞,包装产生慢病毒,感染子宫内膜癌细胞株Ishikawa,观察慢病毒表达载体所携带的GFP基因在细胞内的表达,使用RT-PCR和western-blot检测PIEN基因在细胞内的表达水平.结果:PCR和测序证实,成功克隆慢病毒载体PLIG-PTEN,包装慢病毒,感染真核细胞Ishikawa后48 h,观察到绿色荧光的广泛表述并检测出宿主细胞内表达的PTENmRNA成功表达PTEN蛋白.结论:本实验成功地构建了人PTEN基因慢病毒载体,并观察到PTEN基因在真核细胞中的成功表达.     

携带人VE-cadherin基因的慢病毒载体的构建及其在Sup-B15白血病细胞株的表达

本研究旨在构建携带人血管内皮钙黏蛋白(VE-cadherin)基因的重组慢病毒载体及探讨VE-cadherin蛋白在Sup-B15细胞中的表达。采用RT-PCR扩增人VE-cadherin基因并克隆至pCR-Blunt载体。将VE-cadherinDNA片段连入慢病毒转移质粒pLB,生成重组慢病毒质粒pLB-VEC。用三质粒共转染法包装慢病毒,重组慢病毒感染Sup-B15细胞,在光学显微镜下观察细胞状态,用流式细胞术及Western blot法鉴定VE-cadherin蛋白表达。结果表明,成功扩增出人VE-cadherinDNA片段并克隆至pCR-Blunt载体;亚克隆构建慢病毒载体pLB-VEC。经三质粒包装系统包装后获得高滴度慢病毒颗粒,体外可有效感染Sup-B15细胞。感染后细胞发生明显的形态改变,流式细胞术及Western blot检测到VE-cadherin蛋白表达。结论 :成功构建携带人VE-cadherin基因的慢病毒载体pLB-VEC,并可在Sup-B15白血病细胞株获得有效的表达。     

靶向CD74基因RNA干扰慢病毒稳定感染人胰腺癌细胞株的建立与鉴定

目的构建靶向CD74基因的小发夹RNA慢病毒载体,感染胰腺癌Capan-2细胞株,筛选并建立稳定细胞株。方法应用q RT-PCR法检测不同胰腺癌细胞株间CD74的m RNA表达情况;根据Gene Bank提供的CD74 c DNA序列,设计3条靶向CD74的si RNA序列,构建重组干扰质粒p SUPERretro-puro-CD74-si RNA;将重组干扰质粒转染Capan-2细胞,经过q RT-PCR和Western blot法筛选出最佳干扰效果质粒;慢病毒包装质粒并感染Capan-2细胞株,利用嘌呤霉素筛选,建立稳定低表达CD74细胞株;应用q RT-PCR和Western blot法检测CD74基因m RNA和蛋白的表达抑制率。结果 Capan-2细胞株的CD74 m RNA平均ΔCt值为102.9±6.4,在各胰腺癌细胞株中表达水平最高(P<0.05);携带干扰效果最高si RNA的慢病毒感染Capan-2细胞,建立了稳定低表达CD74基因的Capan-2细胞株,CD74基因m RNA抑制率61%,蛋白抑制率90%。结论成功筛选出靶向CD74的sh RNA最佳干扰质粒,构建了CD74-RNAi慢病毒载体,并稳定感染建立了低表达CD74基因的Capan-2细胞株。     

Biosensors of DsRed as FRET Partner with CFP or GFP for Quantitatively Imaging Induced Activation of Rac, Cdc42 in Living Cells

Purpose

The suboptimal features of the spectral properties of the leading fluorescence resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), limit the full promise of FRET imaging. To overcome the drawbacks, we developed FRET-based, intra-molecular biosensors consisting of CFP/discomona sp red fluorescent protein (DsRed) or green fluorescent protein (GFP)/DsRed as donor/acceptor fluorophores.

Procedures

The biosensors were expressed in NIH3T3 cells. In vitro fluorescence spectroscopy and Rho GTPase activation assays were used to confirm that Rac1 or Cdc42 was activated in serum-starved cells following stimulation with insulin or bradykinin. The transient changes of the amount, location, and translocation of activated Rac1 or Cdc42 in living cells were tracked with confocal imaging.

Results

The increase of FRET efficiency was achieved in the cells expressing the biosensors and was proportional to the levels of activated Rac1 or Cdc42. The localized, transitional, and transient FRET signals were directly and quantitatively imaged with high spatial and temporal resolution. The biosensors were used to analyze and judge the GEF or GAP activities of putative regulatory proteins for Rac1 or Cdc42.

Conclusion

DsRed is a more suitable acceptor in FRET pair with CFP than with GFP in terms of the spectral overlap between the donor and acceptor. The approach can also be applied to many other types of protein behavior in living cells.     

截短型小鼠成纤维细胞生长因子受体-1基因慢病毒载体构建及在真核细胞中的表达

本研究旨在克隆小鼠成纤维细胞生长因子受体1(fibroblast growth factor receptor 1,fgfr1)基因,构建携带增强型绿色荧光蛋白(EGFP)的截短型fgfr1(△fgfr1)重组慢病毒载体并在真核细胞中表达。采用逆转录-聚合酶链反应(RT-PCR)以BALB/c胎鼠脑组织为模板克隆全长型fgfr1基因,连接至克隆载体pCR-Blunt,通过反向PCR技术删除胞内磷酸化区域获得△fgfr1,限制性内切酶酶切后亚克隆至慢病毒转移质粒,构建携带EGFP及△fgfr1双顺反子自身失活型重组慢病毒表达质粒,通过脂质体转染法与包装质粒及包膜蛋白质粒共转染包装细胞293FT,超速离心浓缩病毒颗粒后转染293FT细胞,用荧光显微镜及流式细胞术(FCM)检测EGFP的表达,免疫印迹法(Western blot)鉴定截短型FGFR1蛋白表达。结果表明,成功克隆小鼠fgfr1基因,构建重组慢病毒转移载体LV-IRES-EGFP-△fgfr1及对照载体LV-IRES-EGFP,三质粒系统共转染293FT细胞后获得病毒滴度达到108 TU/ml。以重组病毒载体转染293FT细胞后第4天在荧光显微镜下观察到较强绿色荧光表达,FCM检测转染效率可达95%,Western blot检测显示,转染后293FT细胞表达截短型FGFR1蛋白。结论:成功构建了自身失活型慢病毒载体LV-IRES-EGFP-△fgfr1,并在真核细胞中获得表达。     

Hematopoietic Stem Cell Gene Therapy for the Multisystemic Lysosomal Storage Disorder Cystinosis

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders (LSDs). The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. Cystine accumulates in all tissues and leads to organ damage including end-stage renal disease. Using the Ctns^−/− murine model for cystinosis, we tested the use of hematopoietic stem and progenitor cells (HSPC) genetically modified to express a functional CTNS transgene using a self-inactivating-lentiviral vector (SIN-LV). We showed that transduced cells were capable of decreasing cystine content in all tissues and improved kidney function. Transduced HSPC retained their differentiative capabilities, populating all tissue compartments examined and allowing long-term expression of the transgene. Direct correlation between the levels of lentiviral DNA present in the peripheral blood and the levels present in tissues were demonstrated, which could be useful to follow future patients. Using a new model of cystinosis, the DsRed Ctns^−/− mice, and a LV driving the expression of the fusion protein cystinosin-enhanced green fluorescent protein (eGFP), we showed that cystinosin was transferred from CTNS-expressing cells to Ctns-deficient adjacent cells in vitro and in vivo. This transfer led to cystine decreases in Ctns-deficient cells in vitro. These data suggest that the mechanism of cross-correction is possible in cystinosis.     

Real-time in vivo imaging of stem cells following transgenesis by transposition.

Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.     

Transduction of Human PBMC-Derived Dendritic Cells and Macrophages by an HIV-1-Based Lentiviral Vector System

Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-α. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.     

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34⁺ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34⁺ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34⁺ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45⁺ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.     

慢病毒表达双报告基因载体系统的构建及病毒制备

本研究旨在构建萤火虫荧光素酶(firefly luciferase,FLUC)和红色荧光蛋白(red fluorescent protein,RFP)双基因表达的慢病毒载体,并转染特定细胞系(HeLa),观察双基因的表达。采用PCR技术分别扩增FLUC和RFP报告基因,再将报告基因连接到慢病毒表达载体pLenti-Bi-cistronic中,获得具有双报告基因的慢病毒重组质粒pLenti-FLUC-RFP。然后,用脂质体将该慢病毒重组质粒转染293T细胞,收集纯化慢病毒颗粒。最后,测定病毒滴度,并用所获慢病毒感染HeLa细胞,采用荧光显微镜和荧光素酶报告基因检测试剂盒检测RFP和FLUC的表达。结果表明,酶切和测序证明RFP和FLUC基因成功连入目的载体,所构建的pLenti-FLUC-RFP慢病毒重组质粒序列符合预期。经纯化获得的慢病毒滴度为1×107TU/ml。将pLenti-FLUC-RFP转染HeLa细胞系后,在荧光显微镜下可见大量细胞表达RFP,荧光发光检测仪也检测到转染细胞具有较高的荧光素酶活性。结论:本研究成功构建了具有双报告基因的慢病毒重组质粒pLenti-Ⅲ-FLUC-RFP,经纯化获得了高滴度的慢病毒颗粒,该病毒颗粒具有很好的感染活性,为研究间充质干细胞在体内的动态分布奠定了基础。