Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes.

Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).

Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2.

The blue-green alga, Synechococcus R2, is transformed to antibiotic resistance by chimeric DNA molecules consisting of Synechococcus R2 chromosomal DNA linked to antibiotic-resistance genes from Escherichia coli. Chimeric DNA integrates into the Synechococcus R2 chromosome by homologous recombination. The efficiency of transformation, as well as the stability of integrated foreign DNA, depends on the position of the foreign genes relative to Synechococcus R2 DNA in the chimeric molecule. When the Synechococcus R2 DNA fragment is interrupted by foreign DNA, integration occurs through replacement of chromosomal DNA by homologous chimeric DNA containing the foreign insert; transformation is efficient and the foreign gene is stable. Mutagenesis in some cases attends integration, depending on the site of insertion. Foreign DNA linked to the ends of Synechococcus R2 DNA in a circular molecule, however, integrates less efficiently. Integration results in duplicate copies of Synechococcus R2 DNA flanking the foreign gene and the foreign DNA is unstable. Transformation in Synechococcus R2 can be exploited to modify precisely and extensively the genome of this photosynthetic microorganism.

Length of foreign DNA in chimeric plasmids determines the efficiency of its integration into the chromosome of the cyanobacterium Synechococcus R2.

The photosynthetic cyanobacterium Synechococcus R2 is efficiently transformed by DNA molecules that contain antibiotic genes from Escherichia coli linked to a segment of Synechococcus R2 chromosomal DNA. Antibiotic-resistant transformants result from integration of donor DNA into the cyanobacterial chromosome by homologous recombination. Foreign DNA interrupting the cyanobacterial sequence in the donor molecule integrates by replacement of homologous recipient DNA with donor DNA containing the foreign insert. Foreign DNA linked to the ends of the cyanobacterial fragment in a circular donor molecule integrates by a reciprocal cross-over between donor and recipient sequences. Using donor molecules that contain different lengths of foreign DNA in both of the above positions, we have determined that the probability of integration decreases by half for each increase of 2 to 3 kb in length of a foreign segment, regardless of its position in the donor molecule. The length of one of the two foreign segments has no effect on the integration of the other. Foreign DNA 20 kb in length is completely stable when it has integrated by the replacement mechanism. The ability to stably introduce large pieces of foreign DNA makes Synechococcus R2 an attractive organism in which to study and modify both native and heterologous genes involved in oxygenic photosynthesis.

Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi.

Luciferase (Lux)-encoding sequences are very useful as reporter genes. However, a drawback when applying Vibrio harveyi Lux as a reporter enzyme in eukaryotic cells, is that it is a heterodimeric enzyme, thus requiring simultaneous synthesis of both Lux subunits to be active. To overcome this disadvantage, luxA and luxB genes encoding the A and B subunits of this light-emitting heterodimeric Lux, were fused and expressed in Escherichia coli. Comparative analysis of four fused monomeric Lux enzymes by in vivo enzyme assay, immunoblotting and partial enzyme purification, showed that the fused Lux were active both as AB or as BA monomers, albeit at different levels (up to 80% activity for AB and up to 2% for BA, as compared with the wild type binary A + B construct). One of the LuxAB fusion proteins was stably expressed in calli of Nicotiana tabacum, and displayed very high Lux activity, thus demonstrating its potential as a reporter enzyme in eukaryotic systems.

Genotyping the baboon ABO histo-blood group locus by two-color fluorescence SSCP.

The use of baboons in experimental medicine, especially as organ and tissue donors, would be facilitated by the availability of ABO histo-blood group O animals, which are currently rare. To meet our need in breeding and identifying such animals, we have developed a single-stranded conformational polymorphism (SSCP)-based genotyping assay using fluorescence detection. Various buffers and labeling schemes were evaluated to optimize allele discrimination. Through the use of a nested PCR protocol, single-cell sensitivity was achieved, making the assay applicable to preimplantation diagnosis following in vitro fertilization. We discuss the comparative advantages of SSCP vs. alternative methodologies for genotyping.

Detection of baculovirus gene expression in insect cells and larvae by low light video image analysis.

Recombinant baculovirus isolates BmNPVluc and AcNPVluc (Kopylova-Sviridova et al., 1990) expressing the luc gene in Bombyx mori N-4, and in Sf 9 and Trichoplusia ni 368 cells, respectively, were studied. Luc gene expression driven by baculovirus regulatory elements was detected by enzyme and photometric assays. The expression of recombinant AcNPVluc and BmNPVluc genes in infected larvae of the cabbage looper, T. ni and the tomato hornworm Manduca sexta was analyzed by low-light video image methods. Expression of the luc gene was detected at high levels in both the lepidopteran cells and in third to fifth instar T. ni larvae. However, no light emission was detected in M. sexta caterpillars. High levels of light emission were detected in T. ni larvae when occlusion bodies containing both wild type and recombinant virus were fed to larvae. The results of these experiments demonstrate that video image analysis can be used to monitor the progression of baculovirus infection in susceptible insect cells and larvae. Bioluminescence in recombinant virus infected larvae can be used to determine virus host range, to monitor latent virus infection in insect cells and to assess the spread of recombinant viruses in the environment. Video image analysis was found to be a sensitive method for rapid detection and semiquantitative measurement of luc gene expression in baculovirus infected cells and for monitoring virus infection in larval tissues.

The effect of quick-freezing in ethylene glycol on morphological survival and in vitro development of mouse embryos frozen at different preimplantation stages.

This study was conducted to examine the effect of a quick-freezing protocol on morphological survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages. One-cell embryos were harvested from 6-to 8-wk-old CB6F1 superovulated mice, 20 to 23 h after pairing with males of the same strain and hCG injection. The embryos were cultured in human tubal fluid (HTF) containing 4 mg/ml BSA under mineral oil at 37 degrees C in 5% CO(2) plus 95% room air at maximal humidity. Twenty-four to 96 h after collection, the embryos were removed from culture and frozen at the 2 cell, 4 to 8-cell, compact morula, early blastocyst, expanding blastocyst and expanded blastocyst stages. To perform the quick-freeze procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) + 10 % fetal bovine serum (FBS) + 0.25 M sucrose + 3 M ethylene glycol (freeze medium) for 20 min at room temperature (22 to 26 degrees C) and loaded in a single column of freeze medium into 0.25-ml straws (4 to 5 embryos per straw). The straws were held in liquid nitrogen vapor for 2 min and immersed in liquid nitrogen. Embryos were thawed by gentle agitation in a 37 degrees C water bath for 20 sec and transferred to DPBS + 10 % FBS + 0.5 M sucrose (re-hydration medium) for 10 min at room temperature, rinsed 2 times in HTF plus 4 mg/ml BSA and then cultured for 24 to 96 h. Survival of embryos was based on their general morphological appearance after thawing and their ability to continue development upon subsequent culture in vitro. Survival of blastocysts after thawing also required expansion or reexpansion of the blastocoel after several hours in culture. Significant differences were found in the survival and development of mouse embryos at different developmental stages quick-frozen in ethylene glycol and sucrose: 2-cell embryos 43/84 (51%), 4 to 8-cell embryos 44/94 (47%), morulae and early blastocysts 56/70 (80%; P

Rapid freezing of mouse embryos in ethylene glycol at different preimplantation stages.

The objectives of this study were to examine the effects of a rapid freezing protocol on the survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages, and secondly, to investigate the effect of exposure to 3.0 M EG with 0.25 M sucrose on the survival and in vitro development of mouse embryos without freezing at different developmental stages. To perform the rapid freezing procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) containing 3.0 M EG and 0.25 M sucrose (freeze medium) for 20 min and loaded into 250 microliters straws in a single column of freeze medium. The straws were held in liquid nitrogen (LN2) vapour for 2 min and immersed into LN2. Embryos were thawed in a 37 degrees C water bath for 20 sec and transferred to DPBS supplemented with 0.5 M sucrose (rehydration medium) for 10 min and cultured for 24 to 96 h in HTF (Human Tubal Fluid) plus 4 mg/ml BSA (Bovine Serum Albumin). Significant differences were found in the survival and development of mouse embryos at different developmental stages rapid frozen in EG and sucrose: two cell 43/84 (51%), 4-8 cell 44/94 (47%), morula and early blastocyst 56/70 (80%), expanding and expanded blastocysts 10/59 (17% (p < 0.05). These data indicate that the developmental stage in which mouse embryos are subjected to this quick freeze protocol affects survival and development in vitro and the majority (80%) of morula and early blastocyst stage embryos survive the procedure. No significant differences were observed in the in vitro developmental capacity of embryos at different developmental stages after treatment with high concentrations (3.0 M) of EG solution without freezing. Further investigations are underway to better understand the reasons for different survival rates of embryos frozen at different developmental stages using the present procedure.

Oncolytic vaccinia virus in combination with radiation shows synergistic antitumor efficacy in pancreatic cancer.

Combining oncolytic viruses with conventional therapy such as radiation is an innovative option for pancreatic cancer. We demonstrated that combination of GLV-1h151 and radiation yielded a synergistic cytotoxic effect, with the greatest effect achieved in the AsPC-1cell line. Combination treatment significantly increased apoptosis compared with either single treatment or the control group. In mice bearing human pancreatic tumor xenografts, combination treatment resulted in significantly enhanced inhibition of tumor growth. No evidence of toxicity was observed in mice. These results indicate that the combination of GLV-1h151 and radiation has great potential for translation into clinic practice.

Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in (V600D/E)BRAF mutant melanoma depends on JNK and TNF-α signaling.

Melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. Recent advances in melanoma biology have revealed that RAS-RAF-MEK-ERK signaling has a pivotal role in governing disease progression and treatment resistance. Proof-of-concept clinical studies have shown that direct BRAF inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. Therefore, there is a pressing need to develop new targeted strategies for BRAF mutant melanoma. As such, oncolytic viruses represent a promising cancer-specific approach with significant activity in melanoma. This study investigated interactions between genetically-modified vaccinia virus (GLV-1h68) and radiotherapy in melanoma cell lines with BRAF mutant, Ras mutant or wild-type genotype. Preclinical studies revealed that GLV-1h68 combined with radiotherapy significantly increased cytotoxicity and apoptosis relative to either single agent in (V600D)BRAF/(V600E)BRAF mutant melanoma in vitro and in vivo. The mechanism of enhanced cytotoxicity with GLV-1h68/radiation (RT) was independent of viral replication and due to attenuation of JNK, p38 and ERK MAPK phosphorylation specifically in BRAF mutant cells. Further studies showed that JNK pathway inhibition sensitized BRAF mutant cells to GLV-1h68-mediated cell death, mimicking the effect of RT. GLV-1h68 infection activated MAPK signaling in (V600D)BRAF/(V600E)BRAF mutant cell lines and this was associated with TNF-α secretion which, in turn, provided a prosurvival signal. Combination GLV-1h68/RT (or GLV-1h68/JNK inhibition) caused abrogation of TNF-α secretion. These data provide a strong rationale for combining GLV-1h68 with irradiation in (V600D/E)BRAF mutant tumors.

Enhanced tumor therapy using vaccinia virus strain GLV-1h68 in combination with a ß-galactosidase-activatable prodrug seco-analog of duocarmycin SA.

Breast cancer is the most common cause of cancer-related death worldwide, thus remaining a crucial health problem among women despite advances in conventional therapy. Therefore, new alternative strategies are needed for effective diagnosis and treatment. One approach is the use of oncolytic viruses for gene-directed enzyme prodrug therapy. Here, the lacZ-carrying vaccinia virus (VACV) strain GLV-1h68 was used in combination with a ß-galactosidase-activatable prodrug derived from a seco-analog of the natural antibiotic duocarmycin SA. Tumor cell infection with the VACV strain GLV-1h68 led to production of ß-galactosidase, essential for the conversion of the prodrug to the toxic compound. Furthermore, drug-dependent cell kill and induction of the intrinsic apoptosis pathway in tumor cells was also observed on combination therapy using the prodrug and the GLV-1h68 strain, despite the fact that VACV strains encode antiapoptotic proteins. Moreover, GI-101A breast cancer xenografts were effectively treated by the combination therapy. In conclusion, the combination of a ß-galactosidase-activatable prodrug with a tumor-specific vaccinica virus strain encoding this enzyme, induced apoptosis in cultures of the human GI-101A breast cancer cells, in which a synergistic oncolytic effect was observed. Moreover, in vivo, additional prodrug treatment had beneficial effects on tumor regression in GLV-1h68-treated GI-101A-xenografted mice.

Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent.

Mammary cancers together with cancers of the skin account for about 60% of the total cancers occurring in dogs. The veterinary options for therapeutic management of canine mammary cancer are limited and prognosis for such patients is poor. In this study, we analyzed the functionality of the oncolytic vaccinia virus strain GLV-1h68 as a possible therapeutic agent for canine mammary cancer. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the canine mammary adenoma cell line ZMTH3. Furthermore, after systemic administration this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. The efficient tumor colonization process resulted in inhibition of tumor growth and drastic reduction of tumor size. This is the first report demonstrating that vaccinia virus is an effective tool for the therapy of canine mammary cancers, which might next be applied to dogs with breast tumors.

GroE-mediated folding of bacterial luciferases in vivo.

In this study we present evidence indicating that GroE chaperonins mediate de novo protein folding of heterodimeric and monomeric luciferases under heat shock or sub-heat shock conditions in vivo. The effects of additional groESL and groEL genes on the bioluminescence of Escherichia coli cells expressing different bacterial luciferase genes at various temperatures were directly studied in cells growing in liquid culture. Data indicate that at 42 degrees C GroESL chaperonins are required for the folding of the beta subunit polypeptide of the heterodimeric alpha beta luciferase from the mesophilic bacterium Vibrio harveyi MAV (B392). In contrast, the small number of amino acid substitutions present in the luciferase beta subunit polypeptide from the thermotolerant V. harveyi CTP5 suppresses this requirement for GroE chaperonins, and greatly reduces interaction between the beta subunit polypeptide and GroEL chaperonin. In addition, GroESL are required for the de novo folding at 37 degrees C of a MAV alpha beta luciferase fusion polypeptide that is functional as a monomer. No such requirement for luciferase activity is observed at that temperature with a fusion of the CTP5 alpha and beta subunit polypeptides, although GroE chaperonins can still mediate folding of the CTP5 fusion luciferase. Bacterial luciferases provide a unique system for direct observation of the effects of GroE chaperonins on protein folding and enzyme assembly in living cells. Furthermore, they offer a sensitive and simple assay system for the identification of polypeptide domains required for GroEL protein binding.

Double-stranded gap repair in the photosynthetic prokaryote Synechococcus R2.

The photosynthetic cyanobacterium Synechococcus R2 is transformed by chimeric donor molecules lacking a functional replication origin but containing a region of homology to the recipient chromosome. These integrating donor molecules consist of a fragment of Synechococcus R2 chromosomal DNA cloned in the Escherichia coli vector pBR322 and interrupted by a piece of foreign DNA. During integration, this interrupting DNA is often lost by nonreciprocal exchange between homologous regions of donor and recipient. When transformed with donor molecules containing in vitro-generated double-stranded gaps or deletions as large as 20 kilobase pairs in the fragment homologous to the recipient chromosome, Synechococcus R2 can repair these lesions by using recipient information. Chromosomal DNA of the resulting transformants contains direct repeats of the recipient copy on either side of integrated pBR322 DNA. Homologous recombination between these repeats generates a circular molecule that can be recovered by transformation to E. coli. Plasmids recovered in E. coli contain the entire copy of information initially present in the region of the Synechococcus recipient corresponding to the donor gap or deletion. We suggest applications of this mechanism for cloning of genes identified by transposon mutagenesis.

Structural genes of dinitrogenase and dinitrogenase reductase are transcribed from two separate promoters in the broad host range cowpea Rhizobium strain IRc78.

The nucleotide sequence of the structural gene (nifD) coding for the alpha-subunit of dinitrogenase along with its flanking sequences has been determined in cowpea Rhizobium IRc78. The coding sequence consists of 1500 nucleotides, which corresponds to a predicted amino acid sequence of 500 residues and a molecular weight of 56,025. Nucleotide homology to nifD from the blue-green alga, Anabaena, and Parasponia Rhizobium, are 63% and 90%, respectively. Cowpea Rhizobium IRc78 nifD and nifK (encodes the beta-subunit of dinitrogenase) genes are linked, separated by 69 nucleotides. In contrast to fast-growing rhizobia, the structural genes of dinitrogenase (nifDK) are transcribed from a different promoter than the structural gene of dinitrogenase reductase (nifH). Transcription of nifDK initiates 41 nucleotides upstream of the start codon for the nifDK operon. Two transcription initiation sites, localized at 152 and 114 nucleotides upstream of the start codon, were determined for the nifH operon. Two nucleotide sequences, a hexamer (G-G-T-T-G-C) and a pentamer (T-G-G-C-A), centered at approximately -15 and -25, respectively, are conserved in the nifD and nifH promoter regions and are not present in the 69-nucleotide nifDK junction. No sequence homology other than a possible ribosome binding site, T-T-G-A-[unk]-G-G-A, located 14 nucleotides upstream of the initiation codon was detected between the transcribed but untranslated leader regions of nifD and nifH.

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.