Sirion Biotech | Date: 2013-02-28
The present invention relates to a method for transducing a target cell, the method comprising the step of contacting a target cell with a retroviral vector and a poloxamer having a molecular weight of 12.8 kDa to about 15 kDa. Further, the invention relates to the use of a poloxamer as defined herein, optionally in combination with a polycationic substance as defined herein, for transducing a target cell with a retroviral vector and a kit comprising a retroviral vector, a poloxamer as defined herein and, optionally, instructions for use.
Meyer S.U.,TU Munich |
Thirion C.,SIRION BIOTECH |
Polesskaya A.,University Paris - Sud |
Bauersachs S.,Ludwig Maximilians University of Munich |
And 4 more authors.
Cell Communication and Signaling | Year: 2015
Background: Elevated levels of the inflammatory cytokine TNF-α are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-α and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-α or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation. Results: In this study, we analyzed the impact of TNF-α or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance. Conclusions: The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1. This study indicates thatmiRNAs aremediators of the inhibitory effect of TNF-α on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-α by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-α or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets. © 2015 Meyer et al.; licensee BioMed Central. Source
Hochheimer A.,BRAIN AG |
Hochheimer A.,Amgen Research GmbH |
Krohn M.,BRAIN AG |
Rudert K.,BRAIN AG |
And 4 more authors.
Chemical Senses | Year: 2014
Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities. © The Author 2014. Published by Oxford University Press. Source
Meyer S.U.,TU Munich |
Meyer S.U.,Ludwig Maximilians University of Munich |
Kaiser S.,Ludwig Maximilians University of Munich |
Wagner C.,IMGM Laboratories GmbH |
And 2 more authors.
PLoS ONE | Year: 2012
Background: Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. Methodology/Principal Findings: We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. Conclusions/Significance: We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments. © 2012 Meyer et al. Source
Ruzsics Z.,Max Von Pettenkofer Institute |
Lemnitzer F.,Max Von Pettenkofer Institute |
Thirion C.,SIRION BIOTECH
Methods in Molecular Biology | Year: 2014
Bacterial artificial chromosomes (BACs) are recombinant DNA molecules designed for propagation of large and instable foreign DNA fragment in Escherichia coli. BACs are used in genetics of large DNA viruses such as herpes and baculoviruses for propagation and manipulation of complex genomic regions or even entire viral genomes in one piece. Viral genomes in BACs are ready for the advanced tools of E. coli genetics. These techniques based on homologous or site-specific recombination allow engineering of virtually any kind of genetic changes. In the recent years, BAC technology was also adapted to manipulation of adenovirus genomes and became an effective alternative to traditional genetic engineering of recombinant adenoviruses. © 2014 Springer Science+Business Media, LLC. Source