Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.     

Plaque size phenotype as a selectable marker to generate vaccinia virus recombinants.

In this report, we provide a new method for selection of vaccinia virus recombinants expressing foreign genes. The method is based on the use of the gene encoding the viral 14,000-molecular-weight envelope protein that rescues the small-plaque-size phenotype of a vaccinia virus variant to large-plaque-size virus. Selection of recombinants is easily obtained after visual inspection of large viral plaques.     

A rapid method for screening vaccinia virus recombinants.

A rapid and small-scale method for screening vaccinia virus recombinants employing micrococcal nuclease is described. This protocol utilizes the differential sensitivity of cellular and viral DNA to the nuclease, which can be selectively activated by addition of Ca2+ and inactivated by elimination of Ca2+. Two to five micrograms of viral DNA can be obtained from one infected L cell plate (50 mm) after overnight incubation.     

Puromycin resistance (pac) gene as a selectable marker in vaccinia virus

The antibiotic puromycin, an inhibitor of protein synthesis, was shown to inhibit vaccinia virus (VV) replication. We evaluated the use of puromycin-resistance (pac) gene as a selectable marker in VV. A recombinant vaccinia virus expressing pac (VV-pac) under the control of a viral early/late promoter was constructed and characterized. VV-pac grew in the presence of puromycin at concentrations that were inhibitory for the parental VV and toxic for the cells. Isolation of recombinant VV usually relies on plaque purification under selective conditions. Because virus plaquing was not feasible under inhibitory puromycin concentration, a protocol based on serial passage of virus was devised. The usefulness of this procedure in selecting pac expressing viruses was tested by isolating a recombinant VV.     

Isolation of vaccinia MVA recombinants using the viral F13L gene as the selective marker

Modified vaccinia Ankara (MVA) is a highly attenuated vaccine vector that has an excellent vaccine safety record. Also, as a eukaryotic gene expression vector, MVA can be used in a biosafety level 1 setup, in contrast to more virulent vaccinia virus strains. Isolation of recombinant MVA involves repeated plaquing of the virus and is burdensome because virus plaques are slow to develop and difficult to recognize. To facilitate the generation of MVA recombinants, we have developed a cloning system for MVA based on the selection of the viral F13L gene. Deletion of F13L in MVA produced a small plaque phenotype and a reduction in extracellular virus formation, indicating a severe block in cell-to-cell spread. When using the F13L knockout virus as the parental virus, reintroduction of the F13L gene in the original locus was used as an efficient selection for the isolation of virus recombinants. The selection procedure can be done entirely in the permissive baby hamster kidney (BHK)-21 cell line, does not require plaque isolation, and rendered close to 100% recombinant virus.     

Use of a negative selectable marker for rapid selection of recombinant vaccinia virus

Vaccinia virus has been a powerful tool in molecular biology and vaccine development. The relative ease of inserting and expressing foreign genes combined with its broad host range has made it an attractive antigen delivery system against many heterologous diseases. Many different approaches have been developed to isolate recombinant vaccinia virus generated from homologous recombination; however, most are time-consuming, often requiring a series of passages or specific cell lines. Herein we introduce a rapid method for isolating recombinants using the antibiotic coumermycin and the interferon-associated PKR pathway to select for vaccinia virus recombinants. This method uses a negative selection marker in the form of a fusion protein, GyrB-PKR, consisting of the coumermycin dimerization domain of Escherichia coli gyrase subunit B fused to the catalytic domain of human PKR. Coumermycin-dependent dimerization of this protein results in activation of PKR and the phosphorylation of translation initiation factor, eIF2α. Phosphorylation of this factor leads to an inhibition of protein synthesis, and an inhibition of virus replication. In the presence of coumermycin, recombinants are isolated due to the loss of this coumermycin-sensitive gene by homologous recombination. We demonstrate that this method of selection is highly efficient and requires limited rounds of enrichment to isolate recombinant virus.     

Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants.

Baculovirus and vaccinia virus vectors were used to express the small (S) and medium (M) genome segments of Hantaan virus. Expression of the complete S or M segments yielded proteins electrophoretically indistinguishable from Hantaan virus nucleocapsid protein or envelope glycoproteins (G1 and G2), and expression of portions of the M segment, encoding either G1 or G2 alone, similarly yielded proteins which closely resembled authentic Hantaan virus proteins. The expressed envelope proteins retained all antigenic sites defined by a panel of monoclonal antibodies to Hantaan virus G1 and G2 and elicited antibodies in animals which reacted with authentic viral proteins. A Hantaan virus infectivity challenge model in hamsters was used to assay induction of protective immunity by the recombinant-expressed proteins. Recombinants expressing both G1 and G2 induced higher titer antibody responses than those expressing only G1 or G2 and protected most animals from infection with Hantaan virus. Baculovirus recombinants expressing only nucleocapsid protein also appeared to protect some animals from challenge. Passively transferred neutralizing monoclonal antibodies similarly prevented infection, suggesting that an antibody response alone is sufficient for immunity to Hantaan virus.     

Characterization of polyoma virus early proteins expressed from vaccinia virus recombinants

We previously reported that live recombinant vaccinia viruses (VV) encoding either the large T (LT) or middle T (MT) antigens of polyoma virus (PyV) were able to induce rejection of tumors caused by PyV-transformed cells [Lathe et al., Nature 326 (1987) 878-880]. Here we present evidence that PyV early proteins expressed by the recombinants retain the biochemical characteristics of their authentic counterparts despite the cytopathic effect of VV infection. VV-encoded LT is a nuclear phosphoprotein, with specific DNA binding, ATPase and nucleotide-binding activities. VV-expressed MT associates with cellular kinases, particularly with pp60c-src, by which it is phosphorylated in vitro. Expression levels of LT and MT reached 10(6) molecules per infected cell. The use of VV as a vector is encouraged by the high expression level obtained and because VV infection does not seem to prevent appropriate post-translational processing of proteins encoded by VV recombinants.     

Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.     

The lysopinedehydrogenase gene used as a marker for the selection of octopine crown gall cells

Plant cells transformed into octopine-synthesizing tumour cells by the bacterium Agrobacterium tumefaciens survive when cultured in the presence of homo-arginine (HA), whereas both normal plant cells and nopaline producing plant tumour cells do not. Survival of octopine crown gall cells is due to the activity of the enzyme lysopinedehydrogenase (LpDH) in these cells, which converts toxic homo-arginine into non-toxic homo-octopine. The selective toxicity of homo-arginine for normal cells can be applied for the enrichment of octopine Ti plasmid transformed plant cells vs normal plant cells in mixed cultures.     

Thetms2 gene as a negative selection marker in rice

A conditional negative selection marker is essential for high throughput insertional mutagenesis with any two-element transposon tagging system. Thetms2 gene encodes indoleacetic acid hydrolase (IAAH) which converts naphthaleneacetamide (NAM) to the potent auxin naphthaleneacetic acid, a phytotoxic derivative. This gene, under the control of the manopine synthase gene 2 promoter fromAgrobacterium tumefaciens and exogenously applied NAM, have been used effectively as a negative selector inAc/Ds insertional mutagenesis ofArabidopsis thaliana (Sundaresan et al., 1995). In this study we show thattms2 can also be used as a negative selector in rice. T₁ transgenic seedlings expressing thistms2 gene under the control of themas2’ promoter showed significant reduction in shoot and root growth in the presence of 5–10 μM NAM under specified growth conditions compared to plants not containing this gene.     

Set of vectors for the expression of histidine-tagged proteins in vaccinia virus recombinants

Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells. Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system. To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype. Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E. coli and vaccinia expression plasmids. Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.     

The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana.

The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures. There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium. Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments. Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control. This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.     

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.     

Maize transformation using xylose isomerase gene as a selection marker]

The xylA gene, encoding xylose isomerase, was cloned as a 1342-bp BamHI/SacI fragment from the E. coli. As a selection marker, the xylA gene was fused between the enhanced CaMV 35S promoter (E35S) and terminator (35St) in pBAC413 (Fig.2). pBAC413 was constructed to prevent the expression of sbeIIb in maize. PDS1000/He was used to bombard maize calli, which were induced to form by the elite inbred lines. The selection was carried out on the media containing concentrations of xylose from 0 to 100%. The results showed that the media containing 50% to 100% D-xylose were better, but differed with the genotype of maize (Tables 1 and 2). Successful integration of xylA gene into the maize genome was confirmed by DNA dot blotting, PCR and PCR-Southern hybridization (Figs.4 to 6). A method was established in which transformed maize cells were successively screened on a medium containing xylose instead of antibiotic and herbicide for bio-safety.