|qPCRas a primary screen in drug discovery|
GauravJaggi, Frank Boeckler, Andreas Joerger and Alan Fersht
We report the use of qPCR technique to follow the thermal unfolding of proteins by the binding of the dye SYPRO Orange, and exploit its potential as a robust and high-throughput primary screen for small molecule drug discovery.
|Targeting Inflammatory Cytokines Using Adenoviruses: gene delivery of biological therapies in ovarian cancer|
Michael A. Salako, Hagen Kulbe, Iain A. McNeish, Frances R. Balkwill
Constitutive TNF-alpha expression is characteristic of the malignant ovarian surface epithelium. Adenoviral mutants hold great promise as gene therapy vectors but their efficacy is hindered by an inflammatory cascade orchestrated by TNF-alpha. We found that delivering TNF-alpha shRNA to ovarian cancer cells using oncolytic adenoviruses could reduce the inflammatory cascade generated by adenoviruses and also had direct anti-tumour activity on the cancer cells.
|RNA interference in Manduca sexta induced by double-stranded RNA feeding|
Isabel Gomez, Alejandra Bravo and Mario Soberon
We used a double-stranded RNA feeding approach to silence cadherin-like gene: a protein reported as receptor in several lepidopteran insects for the Cry toxins synthesized by Bacillus thuringiensis. We analized the protein expression from dsRNA-fed and untreated laravae on western blott. M. sexta larvae that ingested dsRNA directed against cadherin exhibited a dramatic suppression of the protein and are resistant to the effect of the Cry1Ab toxin.
|Heterochromatin structure is induced by siRNA targeting HIV-1 promoter region |
K Suzuki1, H Lim1,3, T Ishida2, T Watanabe2, D Cooper1,3, A Kelleher1,3
We transfected siRNA targeting HIV-1 promoter region into a cell line productively infected with HIV-1. ChIP analysis revealed that the RNA duplex induced transcriptional gene silencing and enrichment of Ago1, H3K9me2, and HDAC1 in HIV promoter region to form heterochromatin structure. The data indicates RNA duplex induces the latent phase of HIV infection, since the chromosome formation is very similar to the state of HIV latently infected cell lines
|Efficient downregulation of the lung liquid clearing gammaENaC subunit by RNAi|
Nihal Yueksekdag, Marei Drechsel, Christa Schmidt, and Josef Rosenecker
CF is caused by mutations in the gene encoding for CFTR. CFTR functions as chloride channel on the apical membrane of epithelia thereby regulating the transport of chloride and also sodium ions indirectly. It seems that the regulation of ENaC fails due to the mutated CFTR protein. And it is assumed that ENaC plays a role in the pathogenesis of chronic lung disease in CF-patients.
|In Vitro Potency of Chemically Modified siRNAs Against TNF-alpha|
Sandra M. Ocampo, Anna Aviño, Santiago Grijalvo, José C. Perales and Ramón Eritja
The execution of succesful RNAi experiments depends upon multiple factors: 1. Design and identification of effective and specific siRNA sites 2. Enhancement of pharmacocinetic properties 3. Delivery of siRNA. Therefore we have designed, prepared and functionality tested a variety of chemical modifications. These have been tested in HeLa cells to ascertain wether they influence RNAi inducing activity. We have chosen tumor necrosis factor (TNF-alpha) as a target.
|Critical factors for successful RNAi experiments in primary cells and hard to transfect cell lines|
Markus Zumbansen1, Nicole Spottke1, Sheila Offizier1, Allison St. Amand2, Devin Leake2, Ludger Altrogge1, Meike Weigel1, Sandra Domzalski1, Dietmar Lenz, and Herbert Müller-Hartmann1
The amaxa Nucleofector® Technology is a well established method for effective, non-viral transfection of any nucleic acid substrate into hard-to-transfect cells, especially suspension and primary cells. With the
Nucleofector® 96-well Shuttle® system high throughput applications such as siRNA-library screenings have become amenable for the first time in these cell types. This renders target validation and identification possible
in cell types highly relevant for biomedical research. Here we dis
|Highly Efficient High-Throughput Transfection|
Markus Zumbansen1, Allison St. Amand2, Devin Leake2, Ludger Altrogge1, and Herbert Müller-Hartmann1
Successful RNAi experiments and large-scale siRNA screens require efficient delivery of highly functional and specific nucleic acids including siRNA oligonucleotides, shRNA vectors, or micro RNAs into an appropriate cell system. Cell types relevant for immunological research, such as primary T cells and several suspension cell lines, are poorly accessible using reagent-based transfection approaches.
|Automated High Throughput Nucleofection®|
Claudia Merz, Andreas Schroers, Eric Willimann
Using primary cells for RNAi based applications such as target identification or – validation, requires a highly efficient transfection technology in combination with a reliable and robust automation system. To accomplish these requirements we integrated the amaxa 96-well Shuttle® in a Tecan Freedom EVO® cell transfection workstation which is based on Tecan’s Freedom EVO® liquid handling platform and include all the necessary components and features for unattended cell transfection.