A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-res...     

A quantitative assay for measuring of bovine immunodeficiency virus using a luciferase-based indicator cell line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

Subcellular localization analysis of bovine foamy virus Borf1 protein

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the B...     

多聚TAR-CoreRNA诱饵对人免疫缺陷病毒1型Tat蛋白活性的抑制作用

为了抑制Ｔａｔ蛋白的反式激活作用,在细胞内大量表达外源ＴＡＲＲＮＡ使其与Ｔａｔ蛋白结合,从而竞争性抑制其与ＨＩＶ－１ＬＴＲ的ＴＡＲＲＮＡ元件结合．构建了以ＨＩＶ－１ＬＴＲＹＬ１５８（－１５８～＋１８０）为启动子,分别含有４,８和１５个拷贝的ＴＡＲ－ＣｏｒｅＲＮＡ诱饵（ｄｅｃｏｙ）表达质粒;以荧光酶基因为报告基因,检测了瞬时共转染体系中含不同拷贝数的ＴＡＲ－ＣｏｒｅＤＮＡ转录产物对Ｔａｔ蛋白反式激活作用的影响．结果证明,ＴＡＲ－ＣｏｒｅＲＮＡ诱饵对Ｔａｔ蛋白活性具有很强的抑制作用,其抑制程度与ＴＡＲ－ＣｏｒｅＤＮＡ串联体的拷贝数有关．     

Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2)

Background

Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.

Results

A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-β superfamily members TGF-β 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.

Conclusion

The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.     

人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

HNF1α对人FXR 启动子的调控作用

为探讨肝细胞核因子1α（HNF1α）对人胆汁酸受体（FXR）转录激活的作用及机制,将含HNF1α的真核表达载体（pcDNA3.1(+)HNF1α）和含有FXR启动子的荧光素酶报告基因载体共转染人肝癌细胞系HepG2,检测转染细胞中荧光素酶活性并用半定量RT-PCR、免疫印迹法检测FXR的表达.QuikChange法对FXR启动子HNF1α可能结合位点进行突变,将包含突变点的重组荧光素酶报告质粒单独或与pcDNA3.1(+)共同转染HepG2细胞,检测各组荧光素酶活性.根据凝胶电泳迁移率变化,分析HNF1α与FXR启动子区域的结合.结果发现,转染pcDNA 3.1(+)HNF1α可以上调FXR在HepG2细胞中的表达,并增强FXR启动子活性且具有剂量依赖性;－65～－48区域的点突变,导致FXR启动子活性明显降低,共转染pcDNA3.1(+)HNF1α也不具有增强作用.结果提示,转录因子HNF1α能调控FXR基因表达,其机制为：HNF1α与FXR启动子区域－65～－48区域的反向半位点结合,发挥其反式激活作用.     

牛泡沫病毒BFV3026细胞感染性的确立及包装细胞系的建立

分别将牛泡沫病毒BFV3026接种于胎牛肺细咆(FBL）、牛肺细咆系(BL12)、新生牛肾细胞(NBK)、兔肺细胞(RL)、人乳腺癌上皮细咆(MCF)、293T、HeLa、CV-1、CHO等9种细胞,通过对它们及其传代细胞的病变观察-RT—PCR检测,确立BFV3026对这9种细胞的感染。并以BFV3026原病毒DNA为模板,通过PCR构建以pcDNA3．1(-)为载体的gag—pol、env真核表达质粒共转染BL12细胞,经G418持续筛选,获得8个Neo^R细胞克隆RT—PCR及包装实验证实：其中7个细胞克隆能有效行使包装辅助功能。     

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

The baculovirus–insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell‐surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post‐infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733‐Fc gene were constructed to express GA733‐Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 96 h post‐infection at 0.1 MOI and with GA733‐FcK at 96 h post‐infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 48 h post‐infection at 1 MOI and with GA733‐FcK at 48 h post‐infection at 3 MOI, respectively. These results suggest that the MOI value, post‐infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus–insect cell system.     

FLT-1启动子在血管内皮细胞中特异生物活性的检测及其意义

目的:构建带有组织特异性FLT-1启动子的真核表达载体,检测其在转染的人脐静脉内皮细胞（HUVEC）中对荧光素酶报告基因表达的驱动能力。方法：采用PCR扩增FLT-1启动子,插入到pGL3-Basic-luc载体中,构建携带FLT-1启动子的真核表达载体pGL3-FLT-Basic-luc,经脂质体法转染HUVEC、HepG2、NIH3T3和HEK293细胞,于转染48h后采用双荧光报告系统检测荧光素酶表达活性。 结果:酶切及测序证实构建的pGL3-FLT-Basic-luc载体中含有序列正确的FLT-1基因启动子,双荧光报告系统检测显示,转染的HUVEC细胞其荧光素酶活性明显高于HEK293细胞（P<0.01）,而转染的HepG2和NIH3T3细胞中未检测出荧光素酶表达。结论:克隆的FLT-1启动子具有较高的血管内皮特异性转录活性,可作为血管疾病靶向基因治疗的启动子来源。图     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

In ovo transfection of chicken embryos using cationic liposomes

It is reported that cationic liposomes are capable of transfecting embryos in unincubated fertile chicken eggs and that the cationic liposome, TransfectAce^TM, has superior properties to Lipofectin^TM. In order to determine the duration of expression of genes introduced in this way, embryos were transfected with an expression vector encoding the firefly luciferase cDNA under the control of the Rous sarcoma virus long terminal repeat (LTR). Luciferase activity could be observed consistently in day 3 embryos and activity was detectable up to day 8 of incubation. The relative expression of luciferase under the control of different viral promoters was compared in transfected chicken embryo fibroblasts and day 3 embryos. The cytomegalovirus immediate early promoter and the SV40 early promoter directed the highest amount of expression in fibroblasts while the Rous sarcoma virus LTR caused the highest amount of expression in embryos. Chicken embryo fibroblasts were transfected with the luciferase vector in order to examine duration of reporter gene expressionin vitro. Luciferase expression was decreased exponentially over a 24-day period after which point luciferase activity could no longer be detected. These data suggest that stable integration of transfected DNA using liposomes is a rare event. Nevertheless, liposome-mediated transfection of embryos is suitable for the examination of promoter activityin vivo and may be a useful method to transfect genes to study embryonic development.     

JDV与三种牛反转录病毒相互关系的初步研究

为研究JDV与其它三种牛反转录病毒BIV、BLV、BFV的相互作用关系,将以JDV、BIV、BLV、BFV的LTR为启动子,以Luc为报告基因的质粒和以上病毒反式激活因子的表达质粒共转染BLl2细胞系,通过瞬时表达分析试验证明了JDV和BIV的LTR和Tat之间亲缘关系很近,能够相互激活;JDV Tat可以反式激活BLVLTR,BLVTax不能激活JDVLTR;JDVLTR上存在BFVTas的应答元件;BLV、BFV和BIV的LTR和反式激活因子问不存在相互激活。     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken β-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos. Mol. Reprod. Dev. 49:368–373, 1998. © 1998 Wiley-Liss, Inc.