Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently.The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter.MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells.Cell growth curve showed the Ac-CMV-p35 and Ac-CMV-GFP transduced and non-transduced cells had similar proliferation rate,so baculovirus-mediated p35 expression had no adverse effect on cell proliferation.In addition,baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D,UV or serum-free media.These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.     

Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hyg^r) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hyg^r–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hyg^r stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.     

The histone deacetylase inhibitor sodium butyrate inhibits baculovirus-mediated transgene expression in Sf9 cells

Recent studies have indicated that histone deacetylase inhibitors (HDACis) could enhance and prolong expression of exogenous genes delivered by various viral vehicles in mammalian cells, including baculovirus vectors. In this study, the effects of HDACis on expression of a baculovirus-mediated eGFP reporter gene under control of baculovirus late promoter p10 in Sf9 cells were evaluated. It was found that sodium butyrate (NaBu) decreased the expression level of the target gene driven by p10 promoter by four to fivefold. Moreover, addition of NaBu increased DNaseI-sensitivity of transgene p10 promoter region and did not influence viral DNA replication. FACS assay has shown that both NaBu and fluorodeoxyuridine (FdUrd) blocked Sf9 cells at G1 phase and inhibited the target gene expression. Another HDACi, trichostatin, had little effects on both cell cycle and Ac-p10-eGFP expression, strongly suggesting that cell cycle arrest accounts for the mechanisms by which NaBu inhibits Ac-p10-eGFP expression. The inhibiting effects of NaBu on baculovirus transgene expression in Sf9 cells are promoter specific since the enhancement of NaBu on transgene expression in insect and mammalian cells are mediated by baculovirus harboring a murine cytomegalovirus (mCMV) immediate early promoter. This study was aimed at improving the productivity of the recombinant proteins and providing a better understanding of the epigenetic regulation of baculovirus gene expression.     

Gene transfer of baculoviral p35 by adenoviral vector protects human cerebral neurons from apoptosis

Apoptosis plays an important role in neuronal cell death in both chronic and acute human neurological diseases, including ALS, Huntington's disease, cerebral ischemia, and HIV encephalopathy. We evaluated the ability of an extremely powerful antiapoptotic agent, baculoviral p35, to prevent apoptosis and cell death of human cerebral neurons that undergo severe neurotoxic changes in a culture system when treated with agents that are implicated in human neurological disorders, that is, tumor necrosis factor (TNFalpha) and the HIV proteins Tat and gp120. P35 is a potent broad-spectrum antiapoptotic protein derived from baculovirus, that inhibits nearly all caspases, and has other antiapoptotic actions as well. Adenoviral vectors expressing p35 (Ad. p35) or a control gene (lacZ) efficiently transduced human neurons. Treatment of control cultures with the toxic agents TNFalpha, TNFalpha plus Actinomycin D, or Tat and gp120, induced neurotoxicity and death of neurons. Transduction of neurons with Ad. p35 blocked apoptosis, and eliminated cell death due to TNFalpha, or Tat and gp120. Viral vector transfer of the p35 gene efficiently protects human neurons from TNFalpha, or Tat and gp120-induced apoptosis and cell death. These results suggest that p35 transduction of neurons by viral vectors could be therapeutically useful in the treatment of human neurodegenerative diseases.     

Construction of adenovirus for high level expression of small RNAs in mammalian cells

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.     

Expression of beta‐galactosidase and pig leptin gene in vitro by recombinant adenovirus

Abstract

Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing β‐galactosidase reporter gene (AdCMVLac‐Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac‐Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac‐Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac‐Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac‐Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long‐term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.     

Highly efficient baculovirus-mediated gene transfer into rat chondrocytes

To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.     

Baculovirus-Mediated Gene Expression in Zebrafish

Abstract In an effort to misexpress genes in zebrafish, we tested the ability of baculovirus to infect and drive gene expression in embryos. By injecting virus into specific tissues and using appropriate promoters, both the location and time of gene expression could be controlled. Using a virus with 2 different promoters, LacZ and GFP could be expressed independently. The efficiency of expression appears to depend on the promoter used. As a test of this system, baculovirus was used to ectopically express ephrinB2a in the presomitic mesoderm. EphrinB2a is normally expressed in the posterior region of developing somites, and baculovirus-mediated misexpression caused abnormal somite boundary formation. Baculovirus can thus be used as a tool for gene misexpression experiments in the zebrafish, especially when localized misexpression is required late in development.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

Persistent Baculovirus Infection Results from Deletion of the Apoptotic Suppressor Gene p35

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.     

利用RNA干扰抑制具有多种剪接形式的RNA结合蛋白基因的表达

目的:构建具有多种剪接形式的RNA结合蛋白(RBPMS)基因siRNA的真核表达载体,观察其对RBPMS表达的影响。方法:利用RNA干扰(RNAi)技术,设计并合成了2条针对RBPMS基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上。将重组质粒和带FLAG标签的RBPMS共转染293T人胚肾细胞,通过Western印迹检验RNAi效应。结果:测序证明成功构建了RBPMSsiRNA真核表达载体;Western印迹表明构建的siRNA能有效地抑制RBPMS基因的表达。结论:构建了RBPMSsiRNA的真核表达载体,该siRNA能有效地抑制RBPMS基因的表达。     

杆状病毒作为基因治疗载体的研究进展

杆状病毒是昆虫专一性的病原病毒.近来的研究表明杆状病毒能进入哺乳动物细胞, 但病毒自身不能在哺乳动物细胞中复制, 感染也不引起细胞病变.另外,已经证明杆状病毒能在体外或体内高效地转导许多类型哺乳动物细胞,并且能得到固定表达细胞系,显示了杆状病毒作为基因治疗载体有着良好的应用前景.综述了该领域的最新研究进展并探讨了其发展趋势.     

脆性组氨酸三联体基因小干扰RNA的构建及干扰效果检测

目的:构建脆性组氨酸三联体(FHIT)基因的小干扰RNA(siRNA),并检测其对293T细胞内源性FHIT基因表达的干扰效果。方法:根据FHIT基因序列,通过生物信息学网站预测可能的siRNA,从中筛选出2条合理的FHIT-siRNA,将其克隆到siRNA表达载体pSilencer2.1-U6Hygro中,转化大肠杆菌DH5α,挑取阳性克隆进行测序鉴定;将构建成功的FHIT-siRNA重组载体转染人胚肾细胞293T,Western印迹检测siRNA对293T内源性FHIT表达的干扰效果。结果:构建了2个FHIT-siRNA重组质粒,2条siRNA都有干扰作用,其中一条的作用效果可达50%以上。结论:构建的FHIT-siRNA为进一步研究FHIT的功能提供了有利的工具。     

Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells

Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.     

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.     

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.