Across many areas of biopharmaceutical development, the goal of consistently transfecting appropriate quantities of DNA into cells has often been a significant bottleneck. Electroporation — a method of temporarily permeabilizing cell membranes by using a short electric pulse — has gained ground in recent years as an effective means of transfection. A cell loading system based on electroporation has been designed for ex vivo cell modification in a clinical setting and for incorporation into cGMP processing applicat
Tag: <span>transfection</span>
The non-viral introduction of genes into mammalian cells (transfection) is of growing interest for tissue engineering and as an alternative to the use of viral transfer of recombinant genes. The introduction of a foreign gene into cells in vivo is often limited to the use of viral vectors such as adeno or retroviruses. Viral vector may present several disadvantages or side effects that can be disastrous, and the selection of cells that are transduced by the virus is very poor. A number of non-viral vectors have been explored and used to date: lipid-based carriers, hydrogel polymers, polycationic lipids, polylysine, polyornithine, histones, and other chromosomal proteins, such as hydrogen polymers and precipitated calcium phosphate. Most of these vectors are usable in vitro but are difficult to apply in vivo, especially when local transfection to a specific cell line must be obtained…
G protein-coupled receptors (GPCRs) comprise a “superfamily” of cell surface receptors that play a prominent role in cell signalling and are classified into more than 100 subfamilies according to sequence, ligand structure, and receptor function. They are cell surface receptor proteins with seven transmembrane domains which transduce extracellular signals to the interior of cells through heterotrimeric G proteins. GPCRs’ exposure at the exterior cell surface and strong role in cell regulation has provided a rich target family for small compound therapeutics. Of the estimated 35,000 genes in the human genome, approximately 750 encode for GPCRs; half likely encoding sensory receptors, the remaining half representing potential drug targets. Only about 30 of these potential targets are currently modulated by existing pharmaceuticals with approximately 400 remaining potential pharmaceutical targets for validation…
The Gel Microdrop (GMD) Secretion Assay involves encapsulating cells within a biotinylated agarose matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers. This technology differs from other encapsulation methods in that the small size of the microdrop (<50 ?m diameter) creates a defined microenvironment around the cell without impeding the fusion of nutrients, antibodies, or nucleic acid probes into the GMDs, or the diffusion of secreted products out of the GMDs. Large numbers of GMDs can be readily analyzed using flow cytometry, and sub-populations of rare or high-secreting cells, as small as 0.1%, can be detected and recovered in one day. This assay format is a rapid alternative to limited dilution cloning (LDC)...