Membrane Protein Knockdown Cell Line Development with shRNA
Creative Biolabs can offer membrane protein knockdown cell line development with shRNA 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.
Scientists began utilizing RNA interference (RNAi) to examine gene function after the biological process of RNA interference (RNAi) was first established. shRNAs can be delivered into mammalian cells using plasmid expressing shRNAs transcribed by RNA pol III or modified pol II promoters, and also be given by infecting the cell with virally produced vectors. While siRNA delivers the siRNA duplex to the cytosol, shRNAs can integrate with DNA and consist of two complementary 19-22 bp RNA sequences linked by a short loop of 4-11nt, similar to the hairpin found in naturally occurring miRNA. The shRNA sequence is exported to the cytosol after transcription, where it is identified by an endogenous enzyme called Dicer, which converts the shRNA into siRNA duplexes. This endogenously produced siRNA binds to the target mRNA and is integrated into the RISC complex for target specific mRNA destruction.
Fig.1. Schematic representation of gene-silencing system. (Zhang, 2014)
Applications of Membrane Protein Knockdown Cell Line Development with shRNA
- Down-regulation of a single gene - The knockdown (KD) effect of shRNA clones for a single gene can be studied and compared to that of a scrambled nucleotide control clone.
- Pathway analysis - Genes have been categorized into numerous signal transduction, metabolic, and disease pathways and relationships, as well as gene families and groups.
- Validation studies - For genes/proteins targeted by corresponding shRNA, it can be used for shRNA validation studies and gene/protein functional rescue studies.
Membrane Protein Knockdown Cell Line Development with shRNA from Creative Biolabs includes:
- Construct generation
Synthesis and cloning of shRNA constructs targeting the gene of interest into a suitable mammalian expression vector or lentiviral vector, then production of high-titer lentivirus. We determine the optimal strategy and vector system for you based on your needs.
Fig.2. shRNA expression plasmid. (Tanaka, 2009)
- Selection of stable cell clones
Select stable cell clones after transfection or transduction of target cells.
Fig.3. Detection of shRNA expression. (Zhang, 2014)
- Validation
qRT-PCR - When a shRNA expression cassette targets the matching mRNA, the mRNA is cleaved and degraded, potentially resulting in a drop in protein expression.
Western blot analysis - In most gene KD investigations, protein down-regulation is required, which can be measured by western blot analysis.
Functional assays - The shRNA knockdown effect can be studied by end-point biological and biochemical assay.
Fig.4. Detection of protein expression. (Zhang, 2014)
As an acknowledged biotechnology provider, Creative Biolabs is noted for its first-class services, strictly controlled products and customer-oriented philosophy. Our scientists are able to provide a wide selection of services for both research and commercial use with the best quality at the most competitive price. Please feel free to contact us to confirm how we can be involved in your project.
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References
- Zhang, L., et al. In vivo targeted delivery of CD40 shRNA to mouse intestinal dendritic cells by oral administration of recombinant Sacchromyces cerevisiae. Gene Ther. 2014; 21:709-714.
- Tanaka, H., et al. shRNA expression plasmids generated by a novel method efficiently induce gene-specific knockdown in a silkworm cell line. Molecular biotechnology. 2009; 41(2): 173-179.
