Lars Radke

Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

AbstractQuantitative real-time PCR (qPCR) is commonly used for gene expression analyses with defined documentation guidelines to compare published results. To minimize the impact of variances from qPCR performance, sample handling and processing reference genes are used. Their selection process cannot be completely aligned due to variations in experimental conditions. Furthermore, the named sources of error are also present when determining the stability of the reference genes themselves. Even software applications that are used to identify the best reference genes rarely coincide on their rankings and can be misleading under certain conditions. In previous experiments, peripheral blood mononuclear cells (PBMC) were analyzed to identify the most stable reference gene(s). Twelve of the 13 investigated genes showed sample type specific differences in the expression. Direct mRNA measurement was performed in the form of a NanoString analysis, a multiplexed absolute quantification method. The external validation showed a high concordance of the reference gene expression levels. However, it identified the same sample type specific expression pattern for only some of the tested reference genes. By comparing various combinations of reference genes with both methods we are able to suggest a set of well-performing reference genes. FigureWe here compare the expression of reference genes for qPCR and NanoString data and determine the value of the latter method as a bias-free mRNA quantification method

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans

Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

Miniaturized Flow-Through Bioreactor for Processing and Testing in Pharmacology

Conventional Bioreactor systems for cultivating cells in Life Science have been widely used for decades. An in vitro cell cultivation bioreactor should reliably and reproducibly mimic the in vivo microenvironment of the cultured cells. Normally, mammalian cell cultures are performed in conventional bioreactor devices such as culture flasks and culture-dishes. However, these tools have fundamental limitations due to being inappropriate for high throughput screening and consume a considerable amount of resources and time [1]. Therefore, there is a trend towards miniaturization, disposables and even micro platforms that fulfill increasing demands strongly aiming for production and testing of novel pharmaceutical products. Here we present the development and manufacture of a disposable miniaturized flow-through bioreactor system that can be produced in large numbers at low costs. nanoporous hollow fibers are located at the fluidic sources and drains of the miniaturized bioreactors and retain cells. The necessary mixture of oxygen and carbon dioxide is provided via diffusion through a semi-permeable membrane. Fluidic connections allow the continuous feeding of the cells adding nutrient solution at constant rates at the inlet of the micro bioreactor and removing the solution at the same rate at the outlet. This medium can be collected and used for subsequent analysis. Different designs and concepts for such bioreactors were carried out with varying numbers of plates, and integrated or joined miniaturized reactor chambers. First tests show full technical and biological functionality, cells could successfully be cultivated at high viability rates for some days.

Qualitätsmanagement in der RT-qPCR

Several aspects in the numerous steps of a reverse transcription (RT) quantitative PCR may interfere with the result’s validity. Therefore, before starting the proper investigation, a particular assay establishment is required.