Membrane Protein Knockdown Cell Line Development with siRNA
Creative Biolabs can offer membrane protein knockdown cell line development with siRNA service to meet our customers various requirements. Our technical scientists can not only save the valuable time, but also provide highly stable and qualified products for our clients all over the world.
Small interfering RNA (siRNA) technology, which employs viral vectors, electroporation, and in vivo transfection reagents to inactivate a target gene's expression before it can be translated into protein, has emerged as one of the most promising knockdown technologies for in vitro research. This approach enables the creation of a knock-down line that can be compared to wild type lines in order to determine the biological effects of disrupting the gene of interest's expression. Furthermore, compared to knockouts, siRNA technology allows for a much more cost-effective and efficient method of manufacturing knock-down samples, needing only the most basic lab equipment found in most research labs. Perhaps the greatest advantages of siRNA technology at present are that many pre-existing cell lines can be successfully utilized, and commercially available reagents and siRNA to match almost any defined RNA sequence can be cost effectively purchased.
Fig.1. Mechanism of RNA interference (RNAi). (Mocellin, 2004)
Membrane Protein Knockdown Cell Line Development with shRNA from Creative Biolabs includes:
- siRNA Synthesis Strategies
Several methods for generating siRNA-mediated gene silencing have been devised, each with its own set of benefits and drawbacks. The use of industrial chemical techniques to synthesize, purify, and anneal siRNAs is becoming more common. This process is quick, and the purity is usually rather high. In vitro siRNA synthesis, which uses the T7-phage polymerase, provides an option. Individual siRNA sense and antisense strands are produced by this polymerase, which are then annealed to form siRNAs. RNase digestion and cleaning processes remove extra nucleotides required by the T7 promoter. Otherwise, recombinant Rnase-III can cleave lengthy dsRNAs and generate numerous siRNAs. Although technically simple, this method has the disadvantage of generating nonspecific siRNAs. PolymeraseIII promoter-based DNA plasmids or expression cassettes can be used to make siRNAs.
- siRNA Transfection
Transfection of siRNA into the cells of interest is required to produce RNAi. There are several types of transfection reagents, the most prevalent of which are liposomal or amine-based. Electroporation may be employed in some circumstances, although the risk of cell toxicity is considerable with this method. Various transfection reagents have different reactions in different cell lines, it may be essential to test more than one reagent or technique. Titrating cell density, transfection period, and the ratio of siRNA-to-transfection reagent improves transfection efficiency. The number of cell passages and the application of antibiotics can both affect transfection efficiency.
Creative Biolabs is a well-recognized leader in the field of membrane protein. Based on our advanced technology and high-quality products, our scientists are specialized in membrane protein knockdown cell line development with siRNA.
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Reference
- Mocellin, S., Provenzano, M. RNA interference: learning gene knock-down from cell physiology. J Transl Med. 2004; 2: 39.
