Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

The impact of wine closure and packaging type, and light and temperature exposure on the concentration of 3-alkyl-2 -methoxypyrazines and other key constituents of wine

3-alkyl-2-methoxypyrazines (MPs) are grape- and insect-derived odor-active compounds responsible for vegetative percepts that are detrimental to wine quality when elevated. This study tested both the effect of closure/packaging types and light/temperature storage conditions on MPs (isopropyl-, secbutyl-, and isobutyl-MP) in wine. An MP-emiched wine rapidly (after 140 hours) and significantly decreased in MP concentration after natural and synthetic cork contact (immersion of closures in wine). This decrease was greatest with synthetic closures (70% - 89% reduction) and secbutyl-MP. Subsequently storage trials tested the effects of commercial closure/packaging options (natural cork, agglomerate cork, synthetic corks, screwcaps and TetraPak® cartons) on MPs in MP-emiched Riesling and Cabernet Franc over 18 months. Regardless of packaging, isobutyl-MP was the most altered from bottling. Notably, all MP levels tended to decrease to the greatest extent in TetraPak® cartons (~34% for all MPs) and there was evidence of contribution ofisoproyl- and secbutyl-MP from cork-based closures (i.e. ~30% increase in secbutyl-MP after 6 months) or from an unidentified wine constituent. To test the effects of various light/temperature conditions (light exposed at ambient temperature in three different bottle hues, light excluded at ambient temperature and light excluded at a "cellar" temperature (14°C)), MP-emiched Riesling and Cabernet Franc were also analyzed for MP concentrations over 12 months. MPs did not vary consistently with light or temperature. Other odorants and physico-chemical properties were tested in all wines during storage trials and closely agree with previous literature. These results provide novel insights into MPs during ageing, interactions with packaging and storage conditions, and assist in the selection of storage conditions/packaging for optimal wine quality.

The Terroir of Winter Hardiness: Investigation of Winter Hardiness, Water Metrics, and Yield of Riesling and Cabernet Franc in the Niagara Region Using Geomatic Technologies

Grapevine winter hardiness is a key factor in vineyard success in many cool climate wine regions. Winter hardiness may be governed by a myriad of factors in addition to extreme weather conditions – e.g. soil factors (texture, chemical composition, moisture, drainage), vine water status, and yield– that are unique to each site. It was hypothesized that winter hardiness would be influenced by certain terroir factors , specifically that vines with low water status [more negative leaf water potential (leaf ψ)] would be more winter hardy than vines with high water status (more positive leaf ψ). Twelve different vineyard blocks (six each of Riesling and Cabernet franc) throughout the Niagara Region in Ontario, Canada were chosen. Data were collected during the growing season (soil moisture, leaf ψ), at harvest (yield components, berry composition), and during the winter (bud LT50, bud survival). Interpolation and mapping of the variables was completed using ArcGIS 10.1 (ESRI, Redlands, CA) and statistical analyses (Pearson’s correlation, principal component analysis, multilinear regression) were performed using XLSTAT. Clear spatial trends were observed in each vineyard for soil moisture, leaf ψ, yield components, berry composition, and LT50. Both leaf ψ and berry weight could predict the LT50 value, with strong positive correlations being observed between LT50 and leaf ψ values in eight of the 12 vineyard blocks. In addition, vineyards in different appellations showed many similarities (Niagara Lakeshore, Lincoln Lakeshore, Four Mile Creek, Beamsville Bench). These results suggest that there is a spatial component to winter injury, as with other aspects of terroir, in the Niagara region.

APPLICATION OF GEOMATICS TECHNOLOGIES TO CHARACTERIZE SPATIAL VARIABILITY AT STRATUS VINEYARDS

Vineyards vary over space and time, making geomatics technologies ideally suited to study terroir. This study applied geomatics technologies - GPS, remote sensing and GIS - to characterize the spatial variability at Stratus Vineyards in the Niagara Region. The concept of spatial terroir was used to visualize, monitor and analyze the spatial and temporal variability of variables that influence grape quality. Spatial interpolation and spatial autocorrelation were used to measure the pattern demonstrated by soil moisture, leaf water potential, vine vigour, soil composition and grape composition on two Cabernet Franc blocks and one Chardonnay block. All variables demonstrated some spatial variability within and between the vineyard block and over time. Soil moisture exhibited the most significant spatial clustering and was temporally stable. Geomatics technologies provided valuable spatial information related to the natural spatial variability at Stratus Vineyards and can be used to inform and influence vineyard management decisions.

Students’ Use and Perceptions of Social Networking Technologies: Connections to Reading, Reading Ability, and Self-Perception

Abstract This study was undertaken to examine traditional forms of literacy and the newest form of literacy: technology. Students who have trouble reading traditional forms of literacy tend to have lower self-esteem. This research intended to explore if students with reading difficulties and, therefore, lower self-esteem, could use Social Networking Technologies including text messaging, Facebook, email, blogging, MySpace, or Twitter to help improve their self-esteem, in a field where spelling mistakes and grammatical errors are commonplace, if not encouraged. A collective case study was undertaken based on surveys, individual interviews, and gathered documents from 3 students 9-13 years old. The data collected in this study were analyzed and interpreted using qualitative methods. These cases were individually examined for themes, which were then analyzed across the cases to examine points of convergence and divergence in the data. The research found that students with reading difficulties do not necessarily have poor self-esteem, as prior research has suggested (Carr, Borkowski, & Maxwell, 1991; Feiler, & Logan, 2007; Meece, Wigfield, & Eccles, 1990; Pintirch & DeGroot, 1990; Pintrich & Garcia, 1991). All of the participants who had reading difficulties, were found both through interviews and the CFSEI-3 self-esteem test (Battle, 2002) to have average self-esteem, although their parents all stated that their child felt poorly about their academic abilities. The research also found that using Social Networking Technologies helped improve the self-esteem of the majority of the participants both socially and academically.

How health organizations can apply scenario based planning and analytic technologies to make strategic consequential decisions in a time of uncertainty

The magnitude of the cervical cancer problem, coupled with the potential for prevention with recent technological advances, made it imperative to step back and reassess strategic options for dealing with cervical cancer screening in Kenya. The purpose of this qualitative study was: 1) to explore the extent to which the Participatory Action Research (PAR) methodology and the Scenario Based Planning (SBP) method, with the application of analytics, could enable strategic, consequential, informed decision making, and 2) to determine how influential Kenyan decision makers could apply SBP with analytic tools and techniques to make strategic, consequential decisions regarding the implementation of a Cervical Self Sampling Program (CSSP) in both urban and rural settings. The theoretical paradigm for this study was action research; it was experiential, practical, and action oriented, and resulted in co-created knowledge that influenced study participants’ decision making. Action Africa Help International (AAHI) and Brock University collaborated with Local Decision Influencing Participants (LDIP’s) to develop innovative strategies on how to implement the CSSP. SBP tools, along with traditional approaches to data collection and analysis, were applied to collect, visualize and analyze predominately qualitative data. Outputs from the study included: a) a generic implementation scenario for a CSSP (along with scenarios unique to urban and rural settings), and b) 10 strategic directions and 22 supporting implementation strategies that address the variables of: 1) technical viability, 2) political support, 3) affordability, 4) logistical feasibility, 5) social acceptability, and 6) transformation/sustainability. In addition, study participants’ capacity to effectively engage in predictive/prescriptive strategic decision making was strengthened.