A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1-4 CELLS

The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.     

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes

Abstract Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in R xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from IS to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhe. drovirus from Autographa californica, AcMNPV four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the R xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1、was detected. The R xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.     

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However,the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins(GPs), such as vesicular stomatitis virus G protein(VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64.Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus(AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher p H conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.     

Cloning,expression, and antiviral activity of interferon β from the Chinese microbat,Myotis davidii

Bats are natural reservoir hosts for many viruses that produce no clinical symptoms in bats.Therefore, bats may have evolved effective mechanisms to control viral replication. However, little information is available on bat immune responses to viral infection. Type I interferon(IFN) plays a key role in controlling viral infections. In this study, we report the cloning, expression, and biological activity of interferon β(IFNβ) from the Chinese microbat species, Myotis davidii. We demonstrated the upregulation of IFNB and IFN-stimulated genes in a kidney cell line derived from M. davidii after treatment with poly I:C or infection with Sendai virus. Furthermore, the recombinant IFNβ inhibited vesicular stomatitis virus and bat adenovirus replication in cell lines from two bat species, M. davidii and Rhinolophus sinicus. We provide the first in vitro evidence of IFNβ antiviral activity in microbats, which has important implications for virus interactions with these hosts.     

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.     

Generation of A Stable GFP-reporter Zika Virus System for High-throughput Screening of Zika Virus Inhibitors

Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine(HHT), bruceine D(BD), dihydroartemisinin(DHA) and digitonin(DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549.The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.     

Antisense EGFR sequence reverses the growth properties of human liver carcinoma cell line BEL-7404 in vitro

A recombinant plasmid containing a full length human epidermal growth factor receptor (EGFR) cDNA sequence in antisense orientation was transferred into cells of a human liver carcinoma cell line BEL-7404. Compared with the control cell clone JX-0 transferred with the vector plasmid and the parent BEL-7404 cells, the antisense EGFR transferred cell clone JX-1 showed a decreased EGFR gene expression and reduced significantly the growth potential either in anchorage-dependent or anchorage-independent growth. Furthermore, JX-1 cells appeared to be distinctly dependent on serum concentration for monolayer growth. The results suggested that antisense EGFR could partly block the EGFR gene expression and reverse the malignant growth properties of human liver carcinoma cells in vitro.     

Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 ?-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa’s staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa’s staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells.     

Early Pattern of Epstein-Barr Virus Infection in Gastric Epithelial Cells by “Cell-in-cell”

Epstein-Barr virus(EBV)is an important human dsDNA virus,which has been shown to be associated with several malignancies including about 10%of gastric carcinomas.How EBV enters an epithelial cell has been an interesting project for investigation."Cell-in-cell"infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells.The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction.The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells(GES-1).The infection situation was observed under fluorescence and electron microscopies.Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors.The results demonstrated that EBV could get into gastric epithelial cells by"cell-in-cell"infection but not fully successful due to the host fighting.IL-1β,IL-6 and IL-8 played prominent roles in the cellular response to the infection.The activation of NF-κB and HSP70 was also required for the host antiviral response.The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses.     

Construction and packaging of pseudotype retrovirus containing human N—ras cDNA antisense sequence and its biological effects on human hepatoma cells

N-ras is one of the transforming genes in human hepatic cancer cells.It has been found that N-ras was overexpressed at the mRNA and protein level in hepatoma cells.In order to explore the biological roles of N-ras in human hepatic carcinogenesis and the potential application in control of cancer cell growth,a preudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged.A recombinant retrovirus vector containing antisense or sense sequences of N-ras cDNA was constructed by pZIP-NeoSV(X)1.The pseudotype virus was packaged ang rescued by transfection and infection in PA317 and ψ 2 helper cells.It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PRF/5 hepatoma cells accompanied with inhibition of p21 expression,while the retrovirus containing sense sequence had none.The pseudotype virus had no effect on human diploid fibroblasts.     

A novel system for Agrobacterium-mediated transformation of wheat（ Triticum aestivum L.） cells

A new approach for transforming the cultured cells of wheat (Triticum aestivum L.cv.Ganmai 8)was developed vsing Agrobacterium tumefaciens. The features of the optimum procedure were:(a)both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium(PVA)cells for approximately 16h:(b)the gyratory magnetic field condition was used during cocultivation;(c)the cocultivating period and selecting condition were modified;(d)the recipient cells were at exuberant metabolism and active division while infected with Agrobacterium.Both neomycin phosphotransferase and nopaline synthase assays demonstrated the expression of NPT Ⅱ and NOS genes.located on the T-DNA segment of chimaeric plasmid pGV3850::1103neo.in transformed wheat cell colonies by adopting the techniques of dot blot ndPAGE or high voltage paper electrophoresis,Integration of the foreign genes into wheat genome was confirmed by Southerm blot hybridization.Moreover.a relatively rational method was described for the estimation of transformation frequencies from cultured cell levels.     

Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins.Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer,but these approaches have been largely ineffective;however,a third approach, lentivirus-mediated transgenesis,has successfully produced transgenic livestock.In this study,we generated transgenic(TG) Korean native cattle using perivitelline space injection of viral vectors,which expressed enhanced green fluorescent protein(EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus(FIV) and human immunodeficiency virus(HIV) carrying EGFP were injected into the perivitelline space of MII oocytes.EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group(47.5%±2.2%v.s.22.9%±2.9%).Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers.Ten heifers were successfully impregnated and delivered 10 healthy calves.All of these calves expressed EGFP as detected by in vivo imaging,PCR and Southern blotting.In addition,we established an EGFP-expressing cell line from TG calves,which was followed by nuclear transfer(NT).Recloned 8-cell embryos also expressed EGFP,and there were no differences in the rates of fusion,cleavage and development between cells derived from TG and non-TG calves,which were subsequently used for NT.These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle.Moreover,our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Repfication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 （HIV-1）-based defective lentiviral vectors （DLV） and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV （0.5-1.2 × 107IU/mL）, which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.     

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer.Its mRNA expression was suppressed in most hepatoma samples.In order to study the biological effect of TTR gene on the growth of hepatoma cells,a recombinant vector containing TTR cDNA was constructed by pCMV,then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50% in strength compared with that of the control.This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid.Hepatoma cells of cell lines PLC/PRF/5,SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization.The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721 cells transfected with pCMV alone.However,a remarkable TTR mRNA expression was observed in hepatoma SMMV-7721 cells transfected with pCMV-TTR.It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.     

Insect Cell Culture and Biotechnology

The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.