Murine retroviral vector that induces long-term expression of HIV-1 envelope protein

A retroviral vector was constructed that induces long-term expression of human immunodeficiency virus type 1 (HIV-1) rev, vpu and env genes. The vector contains the neo gene and a cytomegalovirus (CMV) immediate early promoter followed by HIV-1 sequence. When HeLa cells were infected with viral stocks derived from this vector, about 25% of the resulting G418-resistant clones expressed HIV-1 envelope protein (Env), easily detectable by Western blot analysis, metabolic labelling, and syncytium formation after co-cultivation with HeLa-CD4 cells. In most cases the level of Env expression was higher than in a T cell line (H9) chronically infected with HIV-1. Env-expressing HeLa cell lines also expressed Rev, detected by transfection with a Rev-dependent CAT gene construct, and Vpu, detected by immunoprecipitation with a Vpu-specific antiserum. The 75% of G418-resistant HeLa cell lines that did not express Env were found to contain proviruses that had undergone deletion of env sequences corresponding to a known intron; presumably these cell lines arose as a result of infection with virions derived from spliced RNAs. This vector should be useful for studying non-transient effects of HIV Env, Rev and Vpu in tissue culture, and for the production of Env- and/or Rev-expressing cell lines.     

Detection of human T-cell leukaemia virus 1 permissive cells using cell lines producing selectable recombinant virions

A selectable retrovirus vector based on a full length HTLV-1 provirus clone, pCS-HTLV-1, was constructed by replacing the coding regions for tax, rex and the 3′ region of env with the prokaryotic neomycin resistance gene under the control of the CMV promoter. This vector, pHTLV-1-CMVneo, was transfected into HTLV-1 infected human lymphocytes and fibroblasts. The production of recombinant virus by these cells was measured by the transfer of G418 resistance to target cells. Infection of target cells showed a preference for human lymphocytes in addition to two human fibroblast cell lines, Hos7 and RD4, and the African green monkey kidney cell line, Cos7. This system provides a method to study the cellular tropism of HTLV-1 and additionally provides a model to facilitate molecular studies of the natural events of HTLV-1 infection and integration.     

Differentiation of human hematopoietic cells increases expression of a gene transferred by a retroviral vector

To study expression of a retroviral vector in human hematopoietic lineages, two established human hematopoietic cell lines (HL60 and K562) and a human adherent stromal cell line (KM101) were infected with the vector pZIP-SV(X). Expression of the transferred neomycin resistance gene (neor) of pZIP-SV(X) was evaluated as the ability of the cells to form colonies (greater than 50 cells) in an agar assay in the presence of the neomycin analogue, G418. After infection, all three cell lines produced colonies resistant to G418. The level of neor mRNA in separate colonies was analyzed by Northern blot analysis. The neor gene transferred by the vector pZIP-SV(X) was expressed in both human hematopoietic and stromal cell lines. In addition, primary adherent human stromal cells infected with pZIP-SV(X) grew in the presence of G418. To determine if differentiation of hematopoietic cells affects expression of the retroviral vector, HL60 cells infected with pZIP-SV(X) were induced to differentiate, and the level of neor mRNA measured. The amount of neor mRNA increased when HL60 cells were induced to differentiate along the granulocytic pathway. Conversely, when HL60 cells were induced toward monocytoid differentiation (TPA), the level of neor mRNA did not significantly increase. We conclude that the neor gene transferred by a retroviral vector, pZIP-SV(X), is functionally expressed. In addition, expression of the transferred neor gene is regulated during myeloid differentiation of HL60 cells.     

Using simultaneous, tandem gene replacements to study expression of the multicopy ubiquitin-fusion (FUS) gene family of Trypanosoma cruzi

Many genes in trypanosomes exist as members of multicopy gene families. Due to this fact it is frequently difficult to determine if specific members of a gene family are expressed. We describe here a strategy for simultaneous tandem gene replacement in T. cruzi which leads to the replacement of the gene of interest by a silent reporter gene, the expression of which can be assayed in stable transformants. To determine if the FUSI gene (one of 5 copies of the ubiquitin-fusion, FUS, gene family) was expressed, stable G418-resistant transformants were isolated in which the tandemly arrayed CUB2.65 and FUSI genes were precisely replaced by the neomycin phosphotransferase (neo^r) and chloramphenicol acetyltransferase (CAT) genes, respectively. All stable clones carrying the tandem gene replacements were shown to express the CAT activity indicating that FUSI is expressed in mid-log epimastigotes. Northern blot analysis of parasites carrying the tandem gene replacements indicated that at least one other member of the FUS gene family is expressed and that there were no apparent polar effects on the expression of genes downstream of the replacement events. These experiments have demonstrated the utility of tandem gene replacements as a means of inserting a nonselected reporter gene into the chromosome, facilitating the molecular genetic analysis of the expression of multicopy gene families.     

Basic fibroblast growth factor induces c-fos expression in primary cultures of rat striatum

Basic fibroblast growth factor (bFGF) is present in the rat striatum in vivo, where evidence suggests it may have a long-term trophic role in supporting the survival of striatal neurones. To examine the possibility that these effects of bFGF might be mediated by induction of neuronal gene expression, we have investigated the ability of bFGF to stimulate expression of the immediate-early gene c-fos in primary cultures of embryonic rat striatum. The basal levels of c-fos mRNA were low in both neurones and glia in culture. Application of 500 pM bFGF resulted, within 45 min, in a 11-fold increase in the c-fos hybridisation signal in the non-neuronal cells. No significant induction of c-fos mRNA was detected in the striatal neurones at this time. The induction in non-neuronal cells was blocked by the tyrosine kinase inhibitor genistein (100 μM), but not by its inactive structural analogue genistin (100 μM). These results represent a novel mechanism whereby bFGF can exert prolonged effects on striatal function, and indicate that the increases in striatal c-fos gene expression induced by bFGF occur primarily in non-neuronal cells.     

含人组织型纤溶酶原激活剂cDNA重组逆转录病毒的制备…

A recombinant retroviral vector containing tissue-type plasminogen activator (t-PA) cDNA was constructed and transfected into PA317 viral packaging cells, forming intact virus particles. Under electron microscope the recombinant retroviral particles were composed of envelope, capsid and core. These viral particles were spherical with a diameter of 90-180nm, and spread dispersely in the cells. NIH3T3 cell infected by retrovirus particles were screened with G418. The virus titer of 6 x 10(8) CFU/L was verified by counting the positive clones two weeks after screening. The expression of t-PA was demonstrated in the NIH3T3 cells infected with the recombinant virus.     

Specific serodiagnosis of visceral leishmaniasis using a Leishmania donovani antigen identified by expression cloning

A λgt11 expression library was constructed using mRNA from the promastigote form of Leishmania donovani (African isolate MHOM/ET/67/HU3). The library was screened with serum obtained from a patient who contracted visceral leishmaniasis in the Sudan. Several cDNA clones which expressed β-galactosidase/L. donovani antigen fusion proteins were isolated. One of these clones corresponded to a 60 kDa membrane-associated antigen. By a Western blot assay antibodies against the fusion protein were detected universally in the sera of visceral leishmaniasis patients. They were not detected in sera from patients with cutaneous leishmaniasis or other parasitic protozoan infections. The gene coding for this antigen was specific to the genus Leishmania as judged by DNA hybridisation, and its chromosomal location was conserved. A 20 kb mRNA was detected on Northern blots of promastigote RNA.     

逆转录病毒介导的反义c-myb对肝星状细胞增殖及Ⅰ型胶原基因表达的影响

目的:探讨反义c-mybRNA对体外培养的肝星状细胞(HSC)增殖及Ⅰ型胶原mRNA表达的影响。方法:构建含有反向c-myb基因片段的重组逆转录病毒载体pDOR-myb, 将其导入包装细胞PA317中, 收获含病毒的培养上清, 进一步感染体外培养的大鼠HSC, 采用四甲基偶氮唑盐(MTT)法测定细胞增殖反应, 采用半定量RT-PCR检测c-myb、α₁-Ⅰ型胶原mRNA表达。结果:成功分离培养大鼠HSC, 感染pDOR-myb病毒的HSC自身c-myb表达、细胞增殖及α-Ⅰ型胶原mRNA表达显著受抑。结论:c-myb在HSC激活增殖过程中起重要作用, 反义c-myb基因能抑制HSC增殖及Ⅰ型胶原基因表达, 这提示抑制c-myb表达可能是防治肝纤维化的有效途径。     

Avian retroviral expression of luciferase.

Biologically active replication-competent (subgroups A, B, and C) and replication-defective Rous sarcoma virus-derived vectors containing the cDNA encoding firefly luciferase as a reporter gene were constructed. In these retroviral vectors, luciferase is expressed from a spliced subgenomic mRNA. A biologically active replication-defective UR2 virus-derived vector expressing the reporter gene as a gag-luciferase fusion protein from an unspliced genomic mRNA was also constructed. The luciferase reporter gene was used because it lacks homology with chicken genomic sequences and because a rapid and sensitive direct enzymatic assay is available to monitor luciferase expression in retrovirus-infected cells. The levels of luciferase expression in luciferase recombinant retrovirus-infected chicken embryo fibroblasts are greater than 10(3) higher than that detected in uninfected cells or in cells infected with retroviral vectors carrying other genes. Endpoint dilution titration experiments demonstrated that one infected cell can be detected in a background of 10(3) uninfected cells. The vectors are stable in tissue culture and high level expression of the unselected luciferase reporter gene is maintained. The vectors were used to express luciferase in chicken embryos, demonstrating the potential utility of luciferase as a reporter in vivo.     

Stable expression of a selectable myeloproliferative sarcoma virus in murine T lymphocyte and monocyte cell lines

We have investigated whether a retroviral vector based on the myeloproliferative sarcoma virus (MPSV) can be expressed in murine T cells and macrophages. This vector (neoR MPSV) carries the dominant selection marker for neomycin resistance (neoR) and the mos oncogene. The murine T cell line BW5147 and the monocytic cell line P388D1 were either transfected with neoR MPSV DNA or infected with neoR MPSV virus. From both lines, neoR cell clones could be established by retroviral infection, but not by calcium-phosphate precipitation-mediated DNA transfection. The efficiency of infection could be increased 60- to 200-fold upon cocultivation of target cells with irradiated neoR MPSV virus-producing cells. All neoR clones showed neoR MPSV specific sequences as revealed by dot blot and Southern blot analysis. The integration and expression of neoR MPSV was stable over a period of now more than 4 months, even in the absence of selection for neomycin resistance. Northern blot analysis showed that neoR clones express full length neoR MPSV. Further, clones of both T cell and monocyte origin were capable to produce infectious virus particles as revealed by focus formation on fibroblasts and conversion of neomycin sensitive fibroblasts to a neomycin resistant phenotype.     

神经生长因子基因重组逆转录病毒载体的构建与鉴定

目的构建神经生长因子(NGF)基因重组逆转录病毒表达载体,研究NGF在神经干细胞(NSC)中的表达情况。方法从大鼠海马组织提取总RNA,利用RT-PCR的方法获得编码大鼠β-NGF的基因片段,应用基因重组技术,将大鼠β-NGF基因片段克隆到逆转录病毒表达载体pLEGFP-N1中,通过脂质体Lipofectamine2000转染包装细胞PT67,经G418筛选后,收集阳性克隆病毒上清,用于感染神经干细胞(NSC),观察该NSC表达的NGF对PC12细胞突起生长的作用。结果限制性内切酶酶切分析鉴定表明为正确重组子,β-NGF基因在NSC中获得表达,该NSC的培养上清液可以促进PC12细胞突起生长。结论重组逆转录病毒表达载体pLEGFP-NGF构建成功,β-NGF基因可在NSC中表达并具有生物学活性。     

Retroviral interleukin 5 gene transfer into interleukin 5-dependent growing cell lines results in autocrine growth and tumorigenicity

Two interleukin 5 (IL5)-specific retroviral expression vectors have been constructed containing the neomycin gene as selectable marker and either the mouse IL5 cDNA region or the rat genomic IL5 gene under the control of the thymidine kinase promoter. High viral titer supernatants derived from the transfected or infected packaging cell line psi 2 were used to infect the two cell lines B13 and T88M whose growth is dependent on exogenous IL 5. Infection resulted in G418 resistance and IL 5-independent growth with a high frequency. Clones were established which secrete between 2 and greater than 1000 U IL5. The proliferation of the IL5 autocrine growing cells could be inhibited by an antibody directed against the IL5 receptor indicating that they grow as a result of the endogenously produced IL5. Regardless of the amount of IL5 they produced, all of the clones were highly tumorigenic in nucle mice. The phenotype of the tumors was indistinguishable from that of the injected cells. T88M or B13 cells infected with a control virus neither produced IL5, nor became factor independent, nor produced tumors. Together, the IL5 gene transfer and expression into IL5-dependent growing cells are in accordance with the "autocrine growth" hypothesis and contrast analogous experiments with IL4.     

巨细胞病毒对人胚成纤维细胞β肌动蛋白mRNA及微丝的影响

目的：探讨人巨细胞病毒（CMV）对人胚成纤维细胞（HF）的感染及其对细胞肌动蛋白基因 （β-actin）mRNA表达水平和微丝的影响。方法： 应用细胞形态学、RT-PCR半定量法、透射电镜观察CMV对HF细胞的感染及感染后细胞β-actin mRNA和微丝的变化。结果： 经CMV作用后，HF细胞形态由梭形变粗、变圆甚至脱壁；HF细胞有CMV即刻早期抗原基因(IE)mRNA表达，且随着CMV滴度的增加，IE mRNA相对表达量逐渐升高, 而β-actin mRNA相对表达量逐渐减弱，甘油醛3-磷酸脱氢酶基因 （GAPDH） mRNA表达无明显变化。透射电镜下，在HF细胞核、浆内见到大量CMV病毒颗粒；并有细胞微丝断裂、减少，排列紊乱。结论： CMV能感染HF细胞，并在其中活化复制，且致细胞β-actin mRNA表达水平降低；微丝形态、结构发生改变。     

Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene

To increase the selection efficiency and productivity of stable clones expressing recombinant antibody-IL-2 fusion protein, a dicistronic expression vector containing the anti-erbB2 scFv-Fc-IL-2 fusion gene followed by a weakened neo gene, was constructed to allow for the concurrent translation of the recombinant protein and mutated neomycin phosphotransferase from a single mRNA. The presence of the mutant enzyme in the transfectomas resulted in a decreased resistance of cells in the presence of an elevated level of G418 and retarded cell growth. The transfectomas containing the mutant enzyme expressed considerably higher levels of the fusion protein than those containing the normal enzyme. Furthermore, these positive clones had an almost identical level of recombinant gene expression, which was very stable even when the concentration of G418 was significantly increased. Thus, the selection efficiency of strongly positive producers was remarkably increased. Our results demonstrate that the dicistronic expression vector is useful for the selection of highly expressing clones. Combined with an amplification system, this vector may have potential usage for the expression of recombinant antibody and the yield may be further improved by this expression system.     

CD48 May serve as an accessory molecule for the activation of a subset of human γ/δ T cells

To further assess the role of CD48 in the interaction of human γ/δ T cells with their specific target, we generated two series of alloreactive clones, L and K. These clones express a V1-D-J1-C δ chain associated to V3-J2-C2 (L) or V2-J2-C2 (K) γ chain. Functionally they were CTLs able to lyse the sensitizing B-cell line E418. The cytotoxicity of the L and K clones toward E418 was inhibited by anti-CD48 mAb. That of the L clones was also inhibited by anti-HLA class I mAbs. Variation in L and K lysis profile was observed against a panel of CD48 targets, further strengthening the argument that they display distinct specificities and suggesting that they do not recognize CD48. Heterogeneity in TCR gene segment usage, MHC-dependent recognition of E418 by the L clones, and resistance of some CD48⁴ targets strongly suggest that CD48 itself does not interact with L and K TCR. Transfection of CHO cells with CD48 induced killing by the K clones. This killing was inhibited by anti-CD48 mAbs. Taking into account the recent reports on CD48 as an accessory molecule, our results suggest that by binding to CD2 (and/or an unknown ligand), CD48 may serve to strengthen E/T interaction and may contribute to the activation of a minor subset of γ/δ T cells.     

NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.     

小鼠IL－3 cDNA转化细胞移植体内表达的初步研究

随着基因治疗技术的发展和多个细胞因子基因的克隆，人们提出了供细胞因子的基因治疗的设想，以期为肿瘤、老年性痴呆、严重的造血功能障碍等这样一类难治疾病的治疗另辟蹊径。为改善放射损伤小鼠受抑制的造血功能，我们采用基因治疗的方法，将小鼠ＩＬ－３基因ｃＤＮＡ与逆转录病毒载体重组，转染包装细胞ＰＡ３１７，用其分泌的含ＩＬ－３ｃＤＮＡ的复制缺陷逆转录病毒感染、转化ＮＩＨ３Ｔ３细胞，从中筛选可在体外分泌ＩＬ－３的克隆，将可分泌ＩＬ－３的细胞种植到放射损伤小鼠体内后发现：受体小鼠血清中检出了ＩＬ－３的活性；其外周血白细胞计数升至５５万／ｍｍ３，以成熟中性粒细胞为主；肝、脾脏内有大量各个分化阶段的中性粒细胞。这些结果表明：可以利用基因治疗技术，在体内表达ＩＬ－３，改善机体的造血功能。     

Role of the c-fos gene expression on the mitogenic response in EL2 rat fibroblasts

Stimulation of the growth of quiescent fibroblasts by polypeptide growth factors is accompanied by the rapid induction of the c-fos proto-oncogene. To investigate whether there exists a relationship between mitogenic activity and c-fos expression, we analysed cellular responses (DNA synthesis and cell growth) and c-fos gene induction (mRNA and proteins) in a rat embryo fibroblast line (EL2) stimulated with epidermal growth factor (EGF), fibroblast growth factor (FGF), 12-O-tetradodecanoyl phorbol-13-acetate (TPA) and transforming growth factor beta (TGF beta). Our results suggest that the susceptibility of EL2 cells to a growth factor could be predicted as a function of the c-fos expression caused by the same growth factor. These also indicate that the c-fos gene expression may have contributed to moving our cells out of the quiescent state, but it is not the only essential event required to effect EL2 cell growth.     

逆转录病毒介导的人促红细胞生成素基因在淋巴细胞中的表达

本实验设计和构建了人促红细胞生成素（Epo）重组逆转录病毒载体（LXSN-Epo）,载体中小鼠白血病病毒长末端调控序列翻译调控人Epo基因的表达。通过重组逆转录病毒,将Epo基因转导到大鼠T和B淋巴细胞中,DNA-PCR分析证明带有人Epo基因的逆转录病毒已整合到T和B淋巴细胞基因中,RT-PCR分析显示了在两种细胞中均表达了 Epo mRNA。体外生物学活性测定显示,在转导的T淋巴细胞培养上清中Epo表达水平高达243mU/ml,在感染的B淋巴细胞培养上清中Epo活性达43mU/ml。在感染的B细胞中,Epo表达在mRNA和蛋白水平均证明了逆转录病毒能有效地和迅速地转导Epo基因到新培养的B细胞中,而不需要药物选择。这些研究开始了逆转录病毒介导的基因转移走向体内Epo基因治疗研究的过程。