Human immunodeficiency virus vectors for inducible expression of foreign genes.

Tat-dependent expression of an endogenous lethal or deleterious foreign gene might be useful for abrogating the production of human immunodeficiency virus (HIV) from cells. This type of HIV-induced cellular killing, as well as other approaches to gene therapy for HIV infection, would be facilitated by simple HIV vectors that express introduced genes in a Tat-inducible manner. As part of studies to examine the feasibility of this concept, we constructed HIV-1 vectors that express the hygromycin B phosphotransferase gene (Hygr) in a Tat-dependent manner. Comparison of the efficiency of propagation of each vector indicates that sequences extending into the gag open reading frame are necessary in cis for efficient vector propagation. Southern blot analysis of genomic DNA isolated from vector-infected cells demonstrated that the vectors were capable of being propagated as expected without gross rearrangements or deletions. A fragment of the influenza A virus hemagglutinin (H5 HA) gene, capable of eliciting antibody and cytotoxic T-cell responses, was used as a marker for further characterization of the vector system. A Tat-dependent vector conferring the H5 HA+ phenotype was assayed by indirect immunofluorescence, and cells which contained but did not express the H5 HA gene were isolated. The activation of H5 HA expression following HIV infection of Tat- cells that stably contained but did not express the H5 HA construct was determined to be an efficient process.     

A plant virus vector for systemic expression of foreign genes in cereals

Inserts bearing the coding sequences of NPT II and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18-30 days post-inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT-PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS-NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.     

外源基因在植物病毒表达载体中表达策略的研究进展

利用植物病毒表达载体表达外源蛋白是近年来发展起来的具有表达量大,速度快和廉价等优势的生产系统,其有4种构建策略;基因取代,基因插入,融合抗原和基因互补,此外还从病毒表达载体的基础性研究和商业应用方面进行了详细讨论。     

A novel approach for the construction of plant amiRNA expression vectors

Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60 bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60 bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.     

Geminivirus vectors for high-level expression of foreign proteins in plant cells

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.     

A novel block of plant virus movement genes

Hibiscus green spot virus (HGSV) is a recently discovered and so far poorly characterized bacilliform plant virus with a positive‐stranded RNA genome consisting of three RNA species. Here, we demonstrate that the proteins encoded by the ORF2 and ORF3 in HGSV RNA2 are necessary and sufficient to mediate cell‐to‐cell movement of transport‐deficient Potato virus X in Nicotiana benthamiana. These two genes represent a specialized transport module called a ‘binary movement block’ (BMB), and ORF2 and ORF3 are termed BMB1 and BMB2 genes. In agroinfiltrated epidermal cells of N. benthamiana, green fluorescent protein (GFP)‐BMB1 fusion protein was distributed diffusely in the cytoplasm and the nucleus. However, in the presence of BMB2, GFP‐BMB1 was directed to cell wall‐adjacent elongated bodies at the cell periphery, to cell wall‐embedded punctate structures co‐localizing with callose deposits at plasmodesmata, and to cells adjacent to the initially transformed cell. Thus, BMB2 can mediate the transport of BMB1 to and through plasmodesmata. In general, our observations support the idea that cell‐to‐cell trafficking of movement proteins involves an initial delivery to membrane compartments adjacent to plasmodesmata, subsequent entry of the plasmodesmata cavity and, finally, transport to adjacent cells. This process, as an alternative to tubule‐based transport, has most likely evolved independently in triple gene block (TGB), double gene block (DGB), BMB and the single gene‐coded transport system.     

Persistent expression of foreign genes in cultured hepatocytes: expression vectors

We have optimized a liposome-based transfection method that mediated highly efficient stable expression of foreign genes in hepatocytes. Moreover, we have observed that the metallothionein 1 promoter in the bovine papilloma virus-based expression vector drove the highest expression of foreign genes in hepatocytes as compared with the cytomegalovirus and the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoters in the pcDNA 3-based expression vector. The cytomegalovirus promoter failed to yield significant expression in these cells. Furthermore, expression of foreign genes persisted up to at least 15 passages when expression was under the control of either the EF-1alpha or the metallothionein 1 promoter. Thus, these two promoters led to comparable stability of foreign genes in hepatocytes, with the metallothionein 1 promoter yielding a higher level of expression of foreign genes in these cells.     

多基因植物表达载体的构建

将功能互补的抗真菌病基因或抗虫基因共同转化水稻植株,可望获得既抗病又抗虫的转基因水稻植株,马铃薯蛋白酶抑制剂Ⅱ基因PinⅡ,苏云金杆菌毒蛋白CryⅠ(b)基因B.t.,以及PPT乙酰转移酶基因bar构建在一个载体上,水稻碱性几丁质酶基因RC24与大麦核糖体失活蛋白基因B-RIP构建在另一个载体上,两载体共同转化水稻植株的工作正在进行之中。Ⅱ     

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.     

A new set of versatile vectors for the heterologous expression of foreign genes using the baculovirus system

A new set of versatile advanced baculovirus (BV) vectors for the production of fused proteins in insect cells, under the control of the strong polyhedrin promoter of the Autographa california nuclear polyhedrosis virus (AcMNPV), has been constructed. The vectors contain peptide tags which allow immunological detection, as well as purification of recombinant protein produced via the BV expression system     

双价抗虫基因植物表达载体的构建

将蝎毒基因BmKITS和几丁质酶基因chitinase2个抗虫基因采用不同的启动子ubi或35S,连到2个高效的植物表达载体pWM101和pBI101中,2个重组表达质粒分别经过限制性酶切分析和PCR鉴定,实验结果表明2个含有双价抗虫基因的植物重组表达质粒均已构建成功．     

Plasmid vectors containing the tryptophan operon promoter suitable for efficient regulated expression of foreign genes

Derivatives of plasmid pBR322 that are suitable for high-level expression of foreign genes have been constructed. The vectors contain the Escherichia coli tryptophan promoter, the trpE gene, and about 15% of the trpD gene. To obtain expression, foreign genes are fused to the trpD gene fragment. After induction of the trp operon with 3 beta-indolylacrylic acid, trp gene products increase at least 50-fold, to account for 55% of the newly synthesised protein and 30% of total protein in the cell.     

Construction of a novel set of transfer vectors to study vaccinia virus replication and foreign gene expression

Vaccinia virus (VV) is a useful expression vector for many laboratory applications. To date, approximately 60% ( approximately 120) of the VV genes remain uncharacterized. The thought of smallpox being used as a biological weapon has gained attention. In light of this, it is imperative that we continue to study the basic replication of VV, a poxvirus that is closely related to smallpox. A set of plasmid vectors were constructed to generate gene deletions (pZIPPY-NEO/GUS) in VV or for foreign gene expression (pBR-EXPRESS). The vectors contain the Escherichia coli neomycin resistance (neo) and beta-glucuronidase (gusA) genes as selectable markers to facilitate isolation of recombinant viruses. These are the first transfer vectors to use a neo/gusA selection system. We used these vectors to successfully generate a recombinant D9R deletion mutant of VV and to express E. coli lacZ gene. Results indicate that both vectors are highly suited for their designed purpose.     

A Borna disease virus vector for expression of foreign genes in neurons of rodents

An expression cassette for green fluorescent protein was successfully inserted at a site near the 5' end of the genome of Borna disease virus (BDV). When introduced into a mutant virus with highly active polymerase, the foreign gene was strongly expressed in neurons of infected rats. Since BDV can establish long-term persistence in the central nervous system of rodents, it may be used to engineer efficient vectors for specific delivery of foreign genes into highly differentiated neurons.     

Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells

Background: Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. Results: Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. Conclusion: Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins.     

A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.     

A protocol for expression of foreign genes in chloroplasts

Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in approximately 6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.     

Transfer vectors for maximal expression of passenger genes in the Bombyx mori nuclear polyhedrosis virus expression system

A series of Bombyx mori nuclear polyhedrosis virus (Bm-NPV) transfer vectors has been developed containing various lengths of the polyhedrin promoter, including sequences 3' of the initiation codon. The ATG initiation codon was mutated in some of these vectors to allow for the production of authentic nonfusion proteins. The ability of the various polyhedrin promoter constructs to direct expression of foreign gene sequences was assessed using two test genes, chloramphenicol acetyl transferase (cat), and human metallothionein II. Accumulation of cat mRNA and nonfused protein was low when only polyhedrin promoter sequences to -8 (relative to the translational start site of polyhedrin mRNA) were included in the transfer vector, but cat expression was comparable with that of the wild-type polyhedrin gene when promoter sequences to +5 were present. Further addition of polyhedrin gene sequences to +26 or +94 resulted in no further increase in expression. Similar results were obtained for expression of human metallothionein II, where constructs encoding polyhedrin-metallothionein fusion proteins containing polyhedrin sequences to at least +5 resulted in high levels of mRNA and protein accumulation. The expression vectors containing the +5, +26, or +94 BmNPV polyhedrin promoter can thus be used to direct maximal levels of production of nonfused proteins (when the polyedrin ATG has been mutated) or of fusion proteins, depending on which is more suitable for a particular application. These new vectors are a useful addition to those presently available and should increase the utility of the BmNPV expression system for large-scale protein production. (c) 1993 John Wiley & Sons, Inc.     

A novel approach for the construction of multiple shRNA expression vectors

The application of RNA interference (RNAi) as a research and therapeutic tool depends on its ability to silence genes in a sequence-specific manner. Recent studies have reported that the effective knockdown of genes can be achieved by multiple short hairpin RNAs (shRNAs) in a single vector. Moreover, this approach can depress several genes simultaneously. However, current methods for the construction of multiple shRNA vectors often suffer from vector instability and are time-consuming. Here, we describe a simple, quick and low-cost approach to construct a single vector expressing four shRNA sequences driven by four different promoters. Using this vector, we were able to improve the gene silencing efficiency and make it possible to silence four different genes simultaneously, further expanding the application spectrum of RNAi, both in functional studies and therapeutic strategies.