Nervous and nonnervous cell transduction by recombinant adenoviruses that inducibly express the human PrP.

The study of the prion protein (PrP) physiological functions or its specific role in transmissible spongiform encephalopathies (TSE) requires new tools, particularly those able to induce PrP overexpression in a large range of cells, in vivo as well as in vitro. Here we describe the construction of two recombinant adenoviruses encoding the human PrP either with a valine at position 129 (AdTRVal) or a methionine (AdTRMet). Both genes were put under the control of the tetracycline-responsive promoter, allowing tight regulation of PrP expression. AdTRVal and AdTRMet induced high expression of the human PrP in CHO-KI cells and in organotypic brain slices in culture. The proteins expressed from these viruses exhibited a glycosylphosphatidyl inositol (GPI) anchor, proper glycosylation and sensitivity to proteinase K digestion. AdTRVal and AdTRMet will allow future studies on the human PrP and on the role of the codon 129 polyphormism in human TSE.     

Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro.

BACKGROUND: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consuming. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. MATERIALS AND METHODS: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle plasmid containing a transgene and adenovirus genomic DNA. RESULTS: After transfection of Cre-treated DNA into 293 cells, replication-defective viral vectors were rapidly obtained without detectable wild-type virus. CONCLUSION: This system facilitates the development of recombinant adenoviral vectors for basic and clinical research.     

Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses

BACKGROUND: The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. METHODS: Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. RESULTS: The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. CONCLUSIONS: The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.     

Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli.

Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E. coli, by using the bacterial homologous recombination machinery. The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region. All recombinant viral DNAS were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells.     

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.     

Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon.

We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed.     

Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection.

The human T-lymphotropic virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV-I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV-I in rabbits and had a protective effect against HTLV-I infection.     

Plasmid vectors with a semi-synthetic beta-galactosidase gene of E. coli

A partial synthesis of a structural gene for beta-galactosidase and construction of a series of pLZ plasmids for quantitative study of E. coli promoters are reported. The gene was assembled of two short synthetic DNAs and of a 3000 bp long EcoRI fragment (comprising the lacZ sequence 16-3013) isolated from plasmid p198/1 of B. Gronenborn. Among the plasmids constructed, pLZ4 is a promoter-probe vector that contains the semi-synthetic gene fused with a synthetic Shine-Dalgarno sequence and preceded by unique EcoRI and KpnI cleavage sites. On cloning a promoter into these sites, its signal strength in vivo could easily be measured by assaying beta-galactosidase activity. The use of pLZ4 vector was demonstrated by quantifying the effect of T7 early promoters A1 and A2, the latter being found 4,5 times more active under the conditions employed.     

Broad host range and promoter selection vectors for bacteria that interact with plants.

A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed. Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571. They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E. coli (Y. Itoh, J.M. Watson, D. Haas, and T. Leisinger, Plasmid 11:206-220, 1984). Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene. These vectors facilitate the study of gene expression in bacteria under particular environmental conditions. This is illustrated by the expression of the gusA gene under the control of a nod promoter in A. caulinodans nodulating stem-located infection sites on Sesbania rostrata.     

Production of galacto-oligosaccharides by immobilized recombinant beta-galactosidase from Aspergillus candidus

The preparation of galacto-oligosaccharides (GOSs) was studied using the immobilized recombinant beta-galactosidase from Aspergillus candidus CGMCC3.2919. The optimal pH and temperature for the immobilized enzyme were observed at pH 6.5 and 40 degrees C, respectively. Increasing the initial lactose concentration increased the yield of GOSs. The dilution rate was found to be a key factor during the continuous production of GOSs. The maximum productivity, 87 g/L.h was reached when 400 g/L lactose was fed at dilution rate of 0.8/h. The maximum GOS yield reached 37% at dilution rate of 0.5/h. Continuous operation was maintained for 20 days in a packed-bed reactor without apparent decrease in GOS production. The average yield of GOSs was 32%, corresponding to the average productivity of 64 g/L.h, which implied that the immobilized recombinant beta-galactosidase has potential application for GOS production.     

The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation

The versatility of insertional inactivation of β-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including BglII, XhoI, NruI, ClaI, SacI and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites. These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA. The most versatile Polylinker specifies 17 restriction sites in the β-galactosidase α-complementing gene fragment. One of the new polylinkers was inserted into M 13 DNA to produce a vector (M13mIC7) with nine cloning sites.     

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.     

Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase.

A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis. The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA. Upon transformation, they can be used to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type. An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M. maripaludis. The beta-galactosidase gene from Escherichia coli was expressed to approximately 1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M. maripaludis genomic library.     

Efficient transgenesis in farm animals by lentiviral vectors

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.     

Efficient selection of silenced primary cells by flow cytometry.

BACKGROUND: RNA interference has emerged as a new and potent tool to knockdown the expression of target genes and to investigate their functions. For short time experiments with mammalian cell lines, RNA interference is typically induced by transfecting small interfering RNAs (siRNAs). Primary cells constitute important experimental systems in many studies because of their similarity to their in vivo counterparts; however, transfection of these cells has been found to be difficult. As a consequence, RNA interference of primary cells may result in mixed phenotypes because of the simultaneous presence in the same preparation of transfected and nontransfected cells. This may be particularly inconvenient when certain experiments (for example, biochemical analysis) should be performed. METHODS: We use fluorescently labeled siRNAs to induce RNA interference in fibroblasts, and flow-cytometry associated cell sorting to separate subpopulations of transfected cells according to fluorescence intensity. RESULTS: Flow cytometry allows one to discriminate between strongly- and weakly- or nonsilenced fibroblasts, since the fluorescence intensity of transfected cells is related to the number of internalized siRNA copies and to the mRNA knockdown efficiency. CONCLUSIONS: The use of fluorescently labeled siRNAs may allow one to isolate by flow-cytometry associated cell sorting the most efficiently silenced primary cells for subsequent analysis.