Protein interaction assays are a key approach in understanding protein complexes and cellular signaling. Several such interaction assays, including two-hybrid methods, Forster resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC), have been described and expanded in recent years. One challenge that still exists when using these approaches is obtaining equivalent expression of the potential interacting protein pairs, especially in cases where the fusion proteins are expressed from different plasmids. In an attempt to remedy this issue, Christopher Grefen and Michael Blatt from the University of Glasgow report in Rapid Dispatches a simplified methodology that enable the simultaneous cloning of two genes into two different, independent cloning cassettes. The cloning methodology introduced by the authors takes advantage of site-specific recombination wherein each cassette is flanked by a differing attR site and selection element. To demonstrate the utility of their new cloning approach, the authors constructed a vector for improved ratiometric BiFC (rBiFC) analysis. Here, the two genes of interest are cloned simultaneously into the rBiFC vector, which also possesses a RFP gene under control of the same promoter creating an internal marker for expression control. The authors demonstrate the effectiveness of analyzing protein interactions using their rBiFC system by examining interactions between calcineurin B-like (CBL) proteins and the specific serine-threonine kinases these proteins recruit. Although validated by the authors in a plant system and for ratiomeric BiFC, the cloning approach, as well as the rBiFC assay, should prove applicable to other organisms and systems in the future. See “A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC).”
Category: Molecular BiologyStudying gliomas in 3D
The evaluation of cerebral gliomas for malignancy is critical to proper patient survival. Histopathology is mandatory for a definitive diagnosis, but specific immunologic or molecular markers are needed. In a Benchmark article recently published at Rapid Dispatches, a team of researchers led by Lila Kucheryavykh from Universidad Central del Caribe described the use of the fluorescent substrate, 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+), to label glioma cells during the early stages of tumor development. In contrast to the hematoxylin and eosin staining that is often performed when examining tissue slices, ASP+ staining allowed the visualization of 3D structure within living brain slices. In experiments examining the effectiveness of ASP+ staining, glioma cells were implanted into 12-16 week-old mice and staining was performed three weeks post implantation. Imaging of the ASP+ stained tissue allowed reliable 3D visualization of blood vessel configurations and internal tumor cavities for up to two weeks post implantation. The authors did check if ASP+ staining was toxic using a Live/Dead Cytotoxicity kit (it was not), and whether or not the stain also worked with human glioma cells (it did). Taken together, Kucheryavykh et al. demonstrate that ASP+ staining is a simple and effective approach for visualizing gliobastoma tumor cells along with the internal tumor structure and blood vessels, in 3D, in living brain slices during the early stages of tumor development. See “Visualization of implanted GL261 glioma cells in living mouse brain slices using fluorescent 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+).”
Category: Microscopy/ImagingCell patterning: where's the tape?
When it comes to patterning cells on a surface, researchers often take advantage of microfabrication techniques that are capable of producing specific patterns of cells in the micrometer range. The challenge for general users is that such microfabrication approaches also require a clean room along with photolithography equipment and knowledge of fabrication techniques. While non-photolithography methods have been described, many are limited in scope and still require materials not traditionally found in a lab. In a new Benchmark article in Rapid Dispatches, Raquel Perez-Castillejos and her colleagues at the New Jersey Institute of Technology describe their new approach to patterning mammalian cells using adhesive tape. The authors used standard office tape in combination with poly(dimethylsiloxane) to pattern cells with sub-millimeter precision. In a proof-of-concept experiment, the authors performed a wound-healing assay using their tape approach to place cells. This new method, which is being called tape-based soft lithography, should open the technique of cell patterning to a greater number of labs in the years to come. See “Adhesive-tape soft lithography for patterning mammalian cells: application to wound-healing assays.”
Category: Cell Biology