ESACT-UK 15th Annual Meeting
6th-7th January 2005,
Gilbert Murray Conference Centre, University of Leicester
‘Recombinant baculoviruses – more and faster’. Professor Ian Jones, University of Reading
A review of recombinant gene expression using a baculovirus system and methods to optimise this process was presented. The pTriEx-1.1 vector was modified and developed for cloning an open reading frame in a number of different contexts. When a comparison was made between expression of baculovirus proteins in E.coli and baculovirus, one system was always better than the other, depending on the protein. It was shown that one system could rescue the other. Data was presented for work on human kinases, which have been proven to be difficult to express in other systems. Prof. Jones also described work using vectors modified to express cell surface or secreted proteins, focusing on the SARS virus glycoprotein.
‘Production of multiple recombinant baculoviruses using robotic systems’. Dr Kevin Richards, Oxford Brookes University
This presentation began with a description of the development of the flashBAC expression system. The Bacterial Artificial Chromosome (BAC) system was modified to lack ORF 1629, so that only recombinant virus with a restored ORF 1629 can replicate. flashBAC removes the need for plaque purification when producing recombinant virus, thus removing a labour-intensive step of the procedure. This expression system was further developed for high-throughput use in a 24-well plate format by a robotic system. Data presented using this system showed how a high-titre virus stock can be rapidly produced.
‘Analysis of mammalian cell culture processes at the single cell level’. Shanmugapriya Santhalingam, University of Birmingham
This talk described a method for detecting the heterogeneity of a cell population at the single cell level. Data was presented comparing recombinant CHO cell lines cultured in shake flasks and bioreactors. Two methods of analysis were used, standard flow cytometry and the Guava Personal Cell Analysis machine. A positive correlation was found between specific growth rate and mitochondrial activity and also between the biomass of a cell and product concentration.
‘Rapid at-line antibody titre determination using the MININEPHTM endpoint nephelometer’. Leanna Jones, LONZA Biologics Plc
The advantages and disadvantages of the MININEPHä nephelometer over other more traditional methods for measuring antibody concentration were discussed. The MININEPHä was used to determine antibody levels in process fluids from a variety of industrial recombinant cell lines. This system was found to be more rapid and require only small volumes for antibody titre determination.
‘From genome to proteome: alternative splicing as a generator of isoform diversity and substrates for degradation’. Dr Chris Smith, University of Cambridge
An overview of alternative splicing was presented, including data to elucidate the molecular mechanisms behind its regulation. Alternative splicing is important to generate protein diversity, but it is also involved in the quantitative regulation of gene expression at the post-transcriptional level. The mechanism of the Nonsense Mediated Decay (NMD) pathway was described. It was shown that this pathway disposes of mRNAs that harbour premature termination codons, which interact with the exon junction complex to mark mRNA for degradation. Thus the expression of potentially toxic dominant negative proteins produced by aberrant splicing or nonsense mutations is prevented.
‘Functional genomic analysis of genetically modified mouse NS0 cell lines in bioreactor system’. Gary Khoo, University of Birmingham
Using a mouse cDNA microarray, a parental NS0 cell line was compared with a cell line over-expressing Bcl-2 (an anti-apoptotic gene) and a cell line over-expressing p21 (a CDK inhibitor). The data presented demonstrated that the cell line over-expressing Bcl-2 showed metabolic changes indicative of energy diversion to redox homeostasis, resulting in a lower level of transcription and translation.
‘Cell engineering enhances the adaptation of cell lines to serum- and protein-free media’. Paul Clee, University of Birmingham
NS0 and CHO cells were developed to over-express the anti-apoptotic gene Bcl-2. The presentation examined how the over-expression of Bcl-2 eliminated the need for cholesterol and GS supplements during the process of adaptation to serum-free medium. The time taken for adaptation was also reduced and the cell line was found to be more robust in sub-optimal culture conditions.
‘The effect of preventing cell cycle progression through the expression of p21 on the mechanical strength and cellular robustness of CHO cell lines during adaptation to suspension and serum-free growth environments’. Kelly Astley, University of Birmingham
A CHO cell line over-expressing the CDK inhibitor p21 was produced. This cell line could be induced to arrest in the G1 phase of the cell cycle. Arrested cells adapted to serum-free medium and suspension culture more rapidly than wild-type cells. Aggregation of cells, a phenomenon associated with cell stress, was reduced in arrested cells, while cell viability remained high. It was postulated that since cells in G1 are smaller and not dividing, they are less vulnerable to destruction by physical stresses such as stirring.
‘RNA interference, viruses and cancer’. Professor Jo Milner, University of York
This talk started with an introduction to RNA interference (RNAi) and the interferon response within a cell, and then went on to discuss how RNAi can cause apoptosis under stress-free conditions. The Milner lab have utilised RNAi technology to target genes belonging to the human papilloma virus (HPV), which causes cervical cancer. Two genes belonging to the HPV virus, E6 and E7, have been identified and expression of these genes has been silenced using RNAi methods. RNAi against E7 HPV viral mRNA was shown to result in induction of apoptosis in HPV-positive cells only, therefore providing a method for the selective killing of cancer cells. Professor Milner then went on to explain the models used for development of this approach towards the clinic.
‘Production and purification of adenoviral vectors for gene therapy’. Dr Cathy Hogg, Cobra Biomanufacturing plc
This presentation began by outlining the advantages of using perfused stirred tank reactors, as opposed to roller bottles, for the production of adenoviral vectors. Dr Hogg then went on to describe the use of chromatographic purification of the virus, as an alternative to the standard caesium chloride density gradient technique. The advantages of using chromatographic purification, in terms of cost and speed, were then demonstrated.
‘Critical bio-assays for clinical dosing’. Dr Liz Kennings, Cobra Biomanufacturing plc
Bioassays must be performed on a virus product that is intended for use in the clinic, in order to assess identity, quantity, purity and potency. A number of different bioassays were described in detail, including ELISAs, plaque assays and cell killing assays. The need for optimisation and validation, and the control of variability in these bioassays, was also considered.
‘Developments in cell culture perfusion processes and their scale-up to manufacturing’. Dr John Bonham-Carter, Magellan Instruments
Recent developments in cell culture perfusion processes, in particular the alternating tangential flow and filtration system (ATF system), were described. The ATF system utilizes a hollow-fibre filter cartridge and alternating tangential flow to produce filtered product. The benefits of the ATF system were demonstrated, including the production of filtered product, the potential for scale-up and use at high cell densities, and the reduced shear effect on cells.
‘Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification’. Nick Hutchinson, University College London
This presentation discussed the development of laboratory centrifugation studies, which use millilitre quantities of process material to predict the effect of shear stresses on solids in industrial-scale centrifugation. The advantages of such ultra-scale-down models were illustrated.
‘Comparative analysis of HIV-1 recombinant envelope glycoproteins from different culture systems’. Dr Glyn Stacey, National Institute for Biological Standards and Control
The production of HIV-1 recombinant envelope glycoproteins is necessary for vaccine and diagnostics production, structural studies, and the characterisation of immune responses. In this presentation two CHO cell lines, either secreting HIV-1 monomeric (gp120) or oligomeric (gp140) molecular clones, were discussed. Productivity of each of these clones was compared in serum-containing, serum-free and protein-free media. The recombinant glycoprotein products were quantified by ELISA and cell viability, and were also analysed for biological functionality by CD4 and monoclonal antibody binding. Serum-containing media provided the highest level of expression for gp140, whereas expression was greater in serum-free media for gp120. It was shown that protein-free media resulted in misfolding of the gp140 protein, as CD4 binding was lost.
‘Stem cell therapy: challenges for growth control and delivery’. Professor Alicia El Haj, Keele University Medical School
The potential of stem cell therapy for use in regenerative medicine, and the requirement for cells to be delivered and controlled in vitro and in vivo, was highlighted in the introductory comments of this presentation. Professor El Haj then went on to describe the new technologies being developed to grow stem cells in vitro. Tissues may be grown using 3D scaffolds, which may incorporate RGD motifs, hyaluron or collagen coatings. Also, technologies are being developed that use novel bioreactors in the production of tissue-engineered constructs. It is also possible to construct new tissues from stem cells in vivo. A number of methods that are being utilised to monitor tissue formation, including magnetic resonance imaging and optical projection tomography, were discussed.
‘Development of three-dimensional multicellular culture models of pre-invasive breast cancer’. Professor Louise Jones, Barts and the London Hospital
This talk began by highlighting the problems with using conventional culture models when studying ductal carcinoma in situ (DCIS), illustrating the requirement for a multicellular, three-dimensional model of DCIS. Professor Jones described a series of culture model systems, developed to mimic the three-dimensional interactions that occur within DCIS in vivo. Such a model has made it possible to study the mechanisms that promote progression of the carcinoma to invasive disease, and has demonstrated the importance of myoepithelial cells and fibroblasts in tumour invasion. The value of such three-dimensional models in studying phenotypic and genotypic changes that occur prior to invasion was illustrated.
‘Cell cultures as alternatives to animal testing: progressing toward total replacement in the 21st century’ Professor Michael Balls, FRAME
There is increasing pressure for more comprehensive testing of chemicals and various other products, in particular testing for endocrine disruptors, reprotoxicity and genotoxicity. The possibility of using recent developments in molecular and cell biology to design new methods of in vitro testing were emphasised in this final talk, and the integrated use of in silico methods in testing was also mentioned. In particular, new mechanistic studies are required to provide greater understanding of pharmacological and toxicological processes. Finally the importance of validation of these new methodologies, to confirm the reliability and relevance of methods for particular purposes, was discussed.