高效绿色荧光蛋白基因标记系统的建立及其初步应用

逆转录病毒载体编码的众多报告分子被广泛用于分析基因转导率、分选或示踪基因修饰靶细胞 ,特别在改进基因转导条件、阐明造血干细胞 (ＨＳＣ)生物学特性和判定白血病复发的细胞来源等方面发挥了重要作用[1,2 ] 。作为新一代的报告分子 ,增强型绿色荧光蛋白 (ＥＧＦＰ)最近已被用于造血细胞的基因转移和示踪肿瘤转移与血管形成等研究[3 ] 。我们曾建立双启动子逆转录病毒载体介导的ＥＧＦＰ基因转移系统 ,但发现转导靶细胞后荧光强度较低[4 ] 。为提高靶细胞ＥＧＦＰ的表达 ,我们构建了仅携带ＥＧＦＰ单基因的逆转录病毒载体Ｇ1ＦＰ ,并在不…     

应用IRES序列研究人FL与Tpo基因在转染HFCL细胞中的表达

目的　探讨应用内部核糖体进入位点（ＩＲＥＳ）序列对逆转录病毒介导的人Ｆｌｔ３ 配基（ＦＬ）与血小板生成素（Ｔｐｏ） 基因在骨髓基质细胞系ＨＦＣＬ中的表达。方法　利用基因重组技术将ＩＲＥＳ序列与ＦＬ和Ｔｐｏ 基因构建到逆转录病毒载体中,脂质体法将重组质粒ｐＬＦＴＳＮ 和空载体ｐＬＸＳＮ转染ＰＡ３１７细胞,经Ｇ４１８ 筛选。用抗性克隆培养上清感染ＨＦＣＬ细胞,ＲＴＰＣＲ和基因组ＤＮＡＰＣＲ分析ｍＲＮＡ的表达及基因组整合情况,ＣＦＵＧＭ 集落法和Ｔｐｏ 依赖株法测定生物学活性。结果　成功构建出含ＩＲＥＳ序列的ＦＬ与Ｔｐｏ 基因逆转录病毒载体,ｍＲＮＡ水平及基因组中整合有ＦＬ与Ｔｐｏ 基因,生物学活性测定表明转染的骨髓基质细胞同时分泌ＦＬ与Ｔｐｏ。结论　ＩＲＥＳ序列调控ＦＬ与Ｔｐｏ 双基因在骨髓基质细胞中同时独立表达,为进一步研究转基因骨髓基质细胞对造血调控的影响奠定了基础。     

内在核糖体进入位点控制多药耐药基因mdr1表达的研究

目的：观察内在核糖体进入位点（ＩＲＥＳ）控制多药耐药基因ｍｄｒ１的表达能力。方法：以帽依赖性Ｈａｍｄｒ１载体为对照，利用ｐＳＸＬＣ／ｐＨａ系统构建依赖脑心肌炎病毒ＩＲＥＳ翻译的逆转录病毒载体ＨａＩＲＥＳｍｄｒ１（ＨａＩｍｄｒ１），脂质体转染法导入鼠源包装细胞ＧＰ＋Ｅ８６并获得长春新碱耐药细胞；用聚合酶链反应（ＰＣＲ）与流式细胞术（ＦＣＭ）检测ｍｄｒ１基因的转移与表达。结果：病毒生产细胞ＧＰ＋Ｅ８６／ＨａＩｍｄｒ１上清中逆转录病毒的滴度为２．０×１０５ｃｆｕ／ｍｌ，对长春新碱、柔红霉素与紫杉醇产生交叉耐药（２４～５２倍），而且具有多药耐药表型。ＰＣＲ证实ｍｄｒ１基因已稳定整合至ＧＰ＋Ｅ８６／ＨａＩｍｄｒ１细胞基因组；ＦＣＭ分析表明ＩＲＥＳ能引导ｍｄｒ１基因翻译成Ｐ糖蛋白而高效表达，程度略低于Ｈａｍｄｒ１对照。结论：ＩＲＥＳ可引导ｍｄｒ１基因进行有效的帽非依赖性翻译，ｍｄｒ１基因在双顺反子载体中可作为显性选择性基因用于基因治疗。     

重组人造血干细胞因子基因在人造血干／祖细胞的转移和表达

目的：拓展造血干细胞因子（ＳＣＦ）的研究和基因治疗途径。方法：利用基因重组技术，构建了编码可溶性人ＳＣＦ基因的逆转录病毒载体ｐＬＸＳＮ－ＳＣＦ，应用脂质体ｌｉｐｏｆｅｃｔｉｎ基因转移法将重组质粒导入Ψ２和ＰＡ３１７病毒包装细胞，经Ｇ４１８选择性培养基筛选，获得产重组病毒的包装细胞ＰＡ３１７／ＳＣＦ，其病毒效价为（２．４～８．５）×１０５ＣＦＵ／ｍｌ，继而感染人造血干／祖细胞。应用多聚酶链反应、ＡＰＡＡＰ免疫组化染色和化学发光－直接酶标法检测人ＳＣＦ基因在细胞中转移和表达。结果：逆转录病毒载体介导的ｒｈＳＣＦ基因在人造血干／祖细胞中获得有效转移和表达。结论：为进一步研究ＳＣＦ的生物学特性及开展干细胞移植等提供了一定的途径。     

携带绿色荧光蛋白基因的逆转录病毒载体的构建及其介导的T细胞基因转移研究

为了构建含绿色荧光蛋白（green fluorescent protein，GFP）基因的逆转录病毒载体和研究逆转录病毒对T细胞的感染能力，利用亚克隆技术将磷酸甘油酸激酶启动子（phosphoglycerate kinase promoter，PGK）基因和GFP全长cDNA插入逆转录病毒载体pLXSN，采用磷酸钙沉淀法将重组载体转染PA317包装细胞，G418筛选出抗性克隆，收集滴度最高的病毒上清感染NIH3T3和T细胞，在倒置荧光显微镜下观察GFP表达情况。结果表明；重组逆转录病毒载体转染PA317包装细胞后，可在荧光显微镜下观察到GFP的表达。G418筛选后，含GFP的逆转录病毒可感染原代培养的T细胞。结论：逆转录病毒载体能够快速、稳定地将外源基因转移至T细胞，可作为介导T细胞基因转移的重要工具。     

应用荧光原位杂交技术筛选bcr基因探针及分析微小残留病

目的　定量检测慢性髓细胞白血病（ Ｃ Ｍ Ｌ） 造血干细胞移植（ Ｈ Ｓ Ｃ Ｔ） 后ｂｃｒ 基因重排。方法　应用荧光原位杂交（ Ｆ Ｉ Ｓ Ｈ） 技术,从 Ｃ Ｅ Ｐ Ｈ 酵母人工染色体（ Ｙ Ａ Ｃ） 文库的３ 个ｂｃｒ 相关候选克隆中筛选探针,并对７ 例造血干细胞移植后病人进行检测,同时进行细胞遗传学和逆转录聚合酶链反应（ Ｒ Ｔ Ｐ Ｃ Ｒ） 分析。结果　确定了定位于２２ｑ１１ ｂｃｒ 基因区域且可在 Ｐｈ 染色体阳性细胞中易位至９ｑ３４的 Ｙ Ａ Ｃ 克隆７６５ Ｅ３ ,应用该 Ｙ Ａ Ｃ Ｄ Ｎ Ａ 探针进行荧光原位杂交,发现７ 例病人中５ 例存在ｂｃｒ 易位阳性细胞（６ ％ ～９８ ％ ） ,常规 Ｇ 显带检出３ 例, Ｐ Ｃ Ｒ 检出６ 例。结论　 Ｆ Ｉ Ｓ Ｈ 可能成为白血病造血干细胞移植后疗效观察和微小残留病定量监测的重要手段。     

扩增人脐血造血干/祖细胞重建SCID小鼠造血的实验研究

目的　探讨Ｆｌｔ３ 配基（ＦＬ） 、Ｔｐｏ、ＳＣＦ等细胞因子组合对人脐血干／ 祖细胞的扩增及自我更新能力的维持作用。方法　用ＦＬ＋ Ｔｐｏ＋ ＳＣＦ＋ＩＬ６ ＋ＩＬ３＋ ＧＣＳＦ组合,体外扩增脐血ＣＤ３４ ＋ 细胞１４天,将其移植给亚致死剂量照射的ＳＣＩＤ小鼠。结果　扩增的脐血造血细胞可顺利植入ＳＣＩＤ小鼠并重建造血,１８ 只小鼠移植后存活６ 周的有８ 只,其骨髓中仍可检测到人的造血细胞,死亡率５６ ％ ,与对照组（ 死亡率１００％） 比较,χ２ ＝１０．０８,Ｐ＜０．０１。结论　ＦＬ＋ Ｔｐｏ＋ ＳＣＦ＋ＩＬ６ ＋ＩＬ３ ＋ ＧＣＳＦ组合在有效扩增造血细胞的同时可保留造血干／祖细胞的自我更新及造血重建能力。     

琼脂糖凝胶电泳溴化乙锭染色与荧光片段自动分析检测IgH基因产物的比较

在Ｂ 细胞发育过程中 ,免疫球蛋白重链 (ＩｇＨ)经历一个复杂的基因重排过程 ,由此产生 3个可变的互补决定区 (ＣＤＲ) ,这 3个决定区由 4个相对保守的框架区 (ＦＲ)分开 ,ＣＤＲⅢＶ Ｎ Ｄ Ｎ Ｊ序列是可用于分子技术研究的特异性标志物。以ＦＲ1、ＦＲ2和ＦＲ3相应引物的ＰＣＲ扩增ＣＤＲⅢ和ＪＨ序列常用于Ｂ细胞瘤的诊断。ＰＣＲ产物的检测方法有许多 ,如琼脂糖凝胶电泳溴化乙锭染色 (ＥＴＢ ＡＧＧＥ)、荧光片段自动分析(ＡＬＦ)、ＳＳＣＰ、杂合性分析等。本研究以新鲜的或冷冻保存的典型血液淋巴瘤病人的骨髓穿刺细胞为材料 ,其中包…     

少精不育症患者精细胞可疑DNA片段的筛查

目的　筛选与少精不育发生相关的新分子标记。方法　利用现代分子生物学的技术： Ｒ Ａ Ｐ Ｄ、 Ｓｏｕｔｈｅｒｎ 印迹分析（ Ｒ Ｆ Ｌ Ｐ） 、 Ｓｏｕｔｈｅｒｎ 杂交、 Ｎｏｒｔｈｅｒｎ 印迹、克隆、亚克隆及测序对精细胞 Ｄ Ｎ Ａ 作综合分析。结果　在从 Ｒ Ａ Ｐ Ｄ 回收的１２ 条可能与少精不育发生相关的 Ｄ Ｎ Ａ 片段中，有１１ 条被克隆到ｐ Ｃ Ａ Ｐｓ 载体上。在这些克隆片段中８０ ％ 的插入片段是重复顺序，少部分是单拷贝序列。其中一重复顺序在精细胞基因组中具 Ｒ Ｆ Ｌ Ｐ 现象，经 Ｎｏｒｔｈｅｒｎ 印迹分析，发现并无杂交带，说明此片段并非某一表达基因。经对此片段的序列结构进行分析，发现其是含“ Ａ Ａ Ｔ”和“ Ｇ Ｃ Ｇ”密码子重复的中度重复顺序，并且内含有一真核生物顺式作用的启动子序列 Ｔ Ａ Ｔ Ａ 盒“ Ｔ Ａ Ｔ Ａ Ａ Ａ Ａ”，和一个真核生物基因调控的组织特异性增强子序列“ Ｔ Ａ Ｇ Ｃ Ａ Ａ Ａ Ｔ”（ 其反向序列为“ Ａ Ｔ Ｔ Ｔ Ｇ Ｃ Ａ Ｔ”） 。经 Ｉ Ｎ Ｔ Ｅ Ｒ Ｎ Ｅ Ｔ 核酸数据库查寻并未发现有此段序列的报道。结论　此具有 Ｒ Ｆ Ｌ Ｐ 现象的中度重复顺序是与精子发生相关的某一基因的表达调控部分，因其易在基因组中发生变异从而影响到精子     

人脐血和骨髓CD_(34)~ 细胞基因转移效率的比较研究

人脐血和骨髓ＣＤ34 细胞在相同实验条件下,基因转移效率是否相同尚未见报道。我们研究比较了干细胞因子(ＳＣＦ),白细胞介素(ＩＬ)3,ＩＬ6等联合应用对人脐血和骨髓ＣＤ34 细胞基因转移效率的差别,为基因治疗的临床应用提供实验依据。材料和方法1　细胞系　Ｇ418抗性包装细胞株ＰＡ317ＧＣＧＰＸＳＮ及检测病毒滴度用细胞株ＮＩＨ3Ｔ3,均由上海东方肝胆外科医院钱其军博士惠赠。2　病毒上清的制备及病毒滴度测定　按文献[1]进行。3　基因转移　脐血9份,来自兰州市妇幼保健院健康产妇;骨髓9份,来自健康供者。将脐血和骨髓用Ｆｉｃｏｌｌ液(…     

Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.     

Long-term tracking of murine hematopoietic cells transduced with a bicistronic retrovirus containing CD24 and EGFP genes

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation. Gene Therapy (2000) 7, 1193-1199.     

Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T lymphocytes

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.     

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.     

Efficient transduction of human lymphocytes and CD34+ cells via human immunodeficiency virus-based gene transfer vectors

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.     

视锥视杆细胞同源盒基因的慢病毒载体的构建

目的 构建视紫红质启动子(rhodopsin,Rho)驱动的、大鼠视锥视杆细胞同源盒基因(cone-rod homeobox gene,CRX)为目的基因的重组慢病毒载体LV-Rho-EGFP/CRX,研究其在体外的转染效率.方法 用PCR法扩增质粒pEGFP-C3/CRX中的EGFP/CRX基因片段,取代慢病毒载体LV-Rho-EGFP中的EGFP片段,构建表达载体LV-Rho-EGFP/CRX,经酶切和双向测序验证.用磷酸钙沉淀法四质粒共转染293T细胞,包装、收集病毒,利用有限稀释法测定病毒滴度.用慢病毒原液感染光感受器细胞来源的人视网膜母细胞瘤细胞株HXO-RB44,观察感染情况.结果 成功构建慢病毒表达载体LV-Rho-EGFP/CRX,产生的慢病毒滴度为2×104 IU/ml,感染48h后,HXO-RB44细胞内可见强弱不等的绿色荧光.结论 重组载体LV-Rho-EGFP/CRX产生的慢病毒能感染光感受器细胞来源的HXO-RB44细胞,但还需提高转染的效率和滴度.     

Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.     

Immunoresponse against the transgene limits hematopoietic engraftment of mice transplanted in utero with virally transduced fetal liver

In utero cell and gene therapies constitute alternative strategies to the postnatal treatment of inherited diseases. Fetal hematopoietic progenitors could be a potential source of donor cells for these strategies. In this study, hematopoietic lineage-negative fetal liver cells from 14.5-day-old fetuses were transduced under different cytokine and culture combinations using a lentiviral vector expressing the enhanced green fluorescent protein (EGFP). When cells were transduced for 6?h in the presence of mSCF, hTPO and FLT3-L in retronectin-coated dishes at a multiplicity of infection of 10 transduction units/cell, up to 70% of granulo-macrophage colony-forming cells expressed the EGFP reporter gene. In utero transplantation experiments revealed that conditions leading to high transduction efficiencies were associated with poor engraftments of syngeneic recipients. Significantly, this effect was associated with the detection of a humoral and cellular immunoresponse against the transgenic protein. Moreover, the humoral response against EGFP was detected not only in in utero transplanted recipients but also in the operated mothers, suggesting the maternal origin of the anti-EGFP immunoresponse. These observations reinforce the necessity of carefully studying the potential immunoresponses in future prenatal gene therapy protocols.     

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。     

Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct Morquio skin fibroblasts in vitro.

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.