FEN—1基因反义阻断细胞株（FL—FEN—1^—）的建立

ＦＥＮ－１是一咱结构特异性核酸酶,它在ＤＮＡ复制和修复过程中都起着重要的作用。将ＦＥＮ－１基因的ＮｃｏＩ－ＢａｍＨＩ片段反向克隆到哺乳动物细胞重组表达质粒ｐＭＡＭｎｅｏＡｍｐ＾－中,得到ＦＥＮ－１反义表达质粒ｐＭＡＭｎｅｏＡｍｐ＾－ＦＮＢ＾－,并转染ＦＬ细胞,经Ｇ４１８筛选后,获得ＦＥＮ－１基因表达被阻断的哺乳动物细胞ＦＬ－ＦＥＮ－１＾－。对细胞生长曲线的测定发现,ＦＬ－ＦＥＮ－１＾－在地塞米松的     

重组腺病毒介导的白细胞介素—12（IL—12）在肝癌细胞中的表达

利用腺病毒表达系统在肝癌细胞中成功地表达了有生物活性的白细胞介素－１２（ＩＬ－１２）。ＩＬ－１２的Ｐ３５和Ｐ４０ ｃＤＮＡ分别克隆到腺病毒载体ｐＡＣＣＭＶ．ｐＬｐＡ,构建ｐＡＣ／Ｐ３５和ｐＡＣ／Ｐ４０表面质粒。与腺病毒重组质粒ｐＪＭ１７共转染２９３细胞,通过基因重组产生ＩＬ－１２Ｐ３５和Ｐ４０重组腺病毒。用重组腺病毒感染肝癌细胞株ＨｅｐＧ２和ＳＭＭＣ７７２１,经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ检测证明,     

腹腔注射重组腺病毒诱导的免疫反应

为研究重组腺病毒接种实验动物后的免疫反应性,利用ＲＴ－Ｐ　Ｃ　Ｒ方法,从ＨＡＶ的ＲＮＡ中克隆了结构蛋白基因插入穿梭质粒ｐＸＣＸ２Ｎｏｔ　Ｉ,通过磷酸钙－ＤＮＡ共　沉淀技术,将复制缺陷型腺病毒载体与线形化的ｐＸＣＸ２－ＣＭＶ－ＨＡＶ共转染２９３细胞。一系列检测方法证明产生了重组腺病毒ｒＡｄＨＡＶ。纯化后的ｒＡｄＨＡＶ滴度为１×１０９ＴＣＩＤ５０／ｍＬ　,腹腔注射免疫昆明种小白鼠后,可诱导产生抗ＨＡＶ　ＩｇＧ和ＨＡＶ中和抗体。复制缺陷型腺病毒　可作为发展基因工程病毒疫苗载体的有效系统。     

hBMP—2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高,细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃ     

反义RNA抑制人黑色素瘤细胞表面粘附分子CD44的表达及抑瘤效应

将细胞表面粘附分子ＣＤ４４Ｓ的ｃＤＮＡ反向插入到真核细胞表达载体ｐＭＡＭｎｅｏ－ＣＡＴ和ＭＭＴＶ－ＬＴＲ启动子下游,构成ＣＤ４４Ｓ的反义ＲＮＡ载体．将其用电击法导入ＣＤ４４＋的人黑色素瘤细胞系ＨＭＭ２３９,转录出的反义ＲＮＡ能不同程度地抑制ＨＭＭ２３９表面ＣＤ４４的表达．ＣＤ４４的表达被抑制后,瘤细胞与透明质酸的结合力下降,细胞的体外生长速率不受影响．将其接种裸鼠皮下,发现其致瘤性明显降低     

hBMP－2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５＇端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２。将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高;细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃＤＮＡ后,细胞分泌产生的ＢＭＰ－２显著增加。小鼠实验发现,在肌肉内用注射法导入ＢＭＰ－２重组质粒后,局部组织内ＢＭＰ－２的ｍＲＮＡ转录水平也明显提高。     

人癌胚抗原-重组痘苗病毒的构建和制备

痘苗病毒的基因组庞大,结构复杂而特殊,不可能将外源基因直接插入它的基因组,必须利用一种特殊的痘苗病毒质粒,才能构建成功重组痘苗病毒．在分析了痘苗病毒质粒ｐＪ１２０〔含有我国天花疫苗－痘苗病毒天坛株７６１的启动子和胸苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,简称ＴＫ基因）,及含有人癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｎｉｃａｎｔｉｇｅｎ,简称ＣＥＡ）ｃＤＮＡ全序列的质粒ｐ９１０２３Ｂ－ｃｅａ－１７结构的基础上,设计出三步法构建了重组疫苗病毒质粒ｐＪ－ＣＥＡ．经酶切及ＰＣＲ鉴定ｐＪ－ＣＥＡ中ＣＥＡｃＤ－ＮＡ的存在,进一步用同源重组方法构建了表达人ＣＥＡ的重组痘苗病毒,并以人体成纤维细胞作为宿主细胞,对ＣＥＡ－重组痘苗病毒进行了大量培养．再次证实痘苗病毒是良好的真核表达载体,可以高效而准确地表达细胞膜糖蛋白ＣＥＡ．     

可随HeLa细胞传代而传递的反义RNA真核表达系统

为研究反义ＲＮＡ表达载体在细胞内的稳定性,构建了一个特异性针对β地中海贫血基因ＩＶＳ－２－６５４Ｃ→Ｔ（β６５４）突变ｍＲＮＡ前体异常剪接位点的反义ＲＮＡ表达载体ｐＣＭＶＡ．ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后,通过ＲＮＡ定量检测反义片段对β６５４ｍＲＮＡ异常剪接的纠正作用;再从转染后传代５次并冰冻保存１年的ＨｅＬａ细胞中回收反义表达载体,转染另外的β６５４ＨｅＬａ细胞,同样检测它对β６５４ｍＲＮＡ异常剪接的纠正作用．结果显示该载体在细胞传代前后均能阻断β６５４异常剪接,部分恢复其正常剪接途径：用回收的ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后,正常剪接的βｍＲＮＡ水平［β／（β＋β＊）］由０．０５上升到处理后１５ｄ的０．４８,而２种对照质粒处理后对这一比值影响不大．表明ｐＣＭＶＡ可在ＨｅＬａ细胞中随着细胞传代而传递下去,并保持结构与功能完整     

猪生长激素基因在杆状病毒载体系统中的表达

通过对猪生长激素（ｐＧＨ）基因的ｃＤＮＡ进行测序,得到ｐＧＨｃＤＮＡ的全序列,并与Ｓｅｅｂｕｒｇ等报道的序列进行了比较和讨论。然后利用具人工合成启动子和多角体蛋白ＸＩＶ启动子的转移载体质粒ｐＳＸＩＶＶＩ＾＋Ｘ３／４构建出含ｐＧＨ基因的重组质粒ｐＸ３／４－ｐＧＨ。将ｐＸ３／４－ｐＧＨ与致死缺失型线性化ＡｃＭＮＰＶ－ＯＣＣ＾－ＤＮＡ共转染Ｓｆ９细胞,构建出既能形成多角体又能表达ｐＧＨ基因的苜蓿丫纹夜蛾     

化学合成的α—降钙素基因相关基因在大肠杆菌和哺乳…

通过化学法合成α－降钙素基因相关肽的基因,该基因５＇端有ＢａｍＨ Ｉ切点和启始密码,３＇端有ＤａｌＩ切点和终止密码。为了表达这一基因,组建表达载体ｐＸＺ６２．这一表达载体具有Ｔｒｅ启动子,含有Ａｐ＾ｒ和ＸｙＬＥ基因。α－降钙素基因相关肽基因与ｐＸＺ６２边接,构建重组质粒ｐＸＺ１０１;并将该基因与质粒ｐＨβＡＰｒ－３Ｐ－ｎｅｏ连接,构建重组质粒ｐＸＺ２０１．将重组质粒分别转化到大肠矸菌和ＨｅＬａ细胞     

端粒酶RNA的反义cDNA对乳腺癌细胞凋亡的影响(英文)

 为了进一步探讨端粒酶RNA(hTR)的反义cDNA对乳腺癌MCF 7细胞凋亡可诱导性的影响 ,构建了能将外源基因整合至细胞基因组的整合型腺病毒载体vAd AAV ,并将hTR全长cDNA反向连接至此载体上 ,获得反义重组腺病毒vAdT AAV .vAd AAV和vAdT AAV分别感染MCF 7细胞后 ,获得两个细胞株MCF 7 vAd AAV和MCF 7 vAdT AAV ,其中MCF 7 vAdT AAV细胞基因组内整合有hTR反义cDNA并能稳定表达 .利用生存曲线、细胞形态学观察、DNA片段分析和流式细胞分析来测定NaBu和无血清DMEM诱导后细胞凋亡的反应性 .通过生存曲线 ,发现NaBu诱导的MCF 7 vAdT AAV细胞比对照组MCF 7和MCF 7 vAd AAV细胞更早出现凋亡 .电镜下 ,NaBu或去血清诱导的MCF 7 vAdT AAV细胞更早出现凋亡形态学指标 .流式细胞分析和DNA片段凝胶电泳实验均显示MCF 7 vAdT AAV细胞对凋亡的抵抗力下降 .研究结果表明 ,端粒酶RNA的反义cDNA使乳腺癌MCF 7细胞的凋亡可诱导性增强     

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.     

Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.

Immortalized cell lines have been used to study infection and replication of adeno-associated virus (AAV) in culture, but primary cells presumably provide a better model for AAV behavior in animals. Here, we have evaluated the ability of AAV vectors to transduce primary and immortalized strains of human epithelial cells and fibroblasts. Two AAV vectors were used, one that transduced an alkaline phosphatase gene (AAV-LAPSN), and one that transduced a beta-galactosidase/neomycin phosphotransferase fusion gene (AAV-L beta geo). The transduction efficiency of the AAV-LAPSN vector, quantitated by measurement of alkaline phosphatase-positive cell foci following infection, was 10 to 60 times greater in immortalized human cells than in primary cells, and total alkaline phosphatase activity in cell lysates was 40 to 50 times greater in immortalized cells. The AAV-L beta geo vector gave similar results. In contrast, the transduction efficiency of a retrovirus vector encoding alkaline phosphatase was equivalent in primary and immortalized cells. Analysis of the quantity and state of the AAV vector genomes in cells showed that primary and immortalized cells contained comparable numbers of vector copies per cell and that the vast majority of vector DNA was not integrated into the cell genome. Additionally, the level of AAV vector-derived message paralleled the transduction efficiency. These results indicate that the block to functional transduction in primary cells occurred after virus entry and limited the abundance of vector-derived message. Data from AAV transduction in cultures of human cells containing immortalizing genes suggest that cellular changes secondary to the introduction of immortalizing genes increased permissiveness for transduction by AAV vectors. In summary, our data demonstrate that AAV vectors transduce primary human cells much less efficiently than immortalized cells and indicate the importance of using primary cells to evaluate AAV vectors for gene therapy applications.     

A High-Capacity, Capsid-Modified Hybrid Adenovirus/Adeno-Associated Virus Vector for Stable Transduction of Human Hematopoietic Cells

To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, DeltaAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The DeltaAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity DeltaAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human gamma-globin gene and the HS2 and HS3 elements of the beta-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with DeltaAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.     

Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.     

腺相关病毒与人多药耐药基因重组载体的构建及在NIH3T3细胞中的表达

腺相关病毒 (adeno- associated virus,AAV)属细小病毒科 ,是一种最小的动物病毒 .具有其他病毒载体所没有的优点 ,在基因治疗中日益受到瞩目 .以 AAV的一种多克隆载体为基础 ,构建了携带 MDR1基因的重组腺相关病毒载体 (r AAV- MDR1 ) ,经 2 93细胞包装成重组病毒 .将重组质粒、重组病毒分别转染和感染 NIH3T3细胞 ,用 PCR和 MTT法检测了人 MDR1基因的转导及表达 .为 MDR1基因用于临床和腺相关病毒载体在基因治疗中的应用提供了依据     

Efficient site-specific integration of large transgenes by an enhanced herpes simplex virus/adeno-associated virus hybrid amplicon vector

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in approximately 22% of transduced cells; of those cells, approximately 70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.     

Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.     

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.