SHRNA Gene Knockdown Stable Cell Line Construction

Seattle Genova provides a series of shRNA stable cell line construction services. 

shRNA is a sequence of RNA which gives rise to a tight hairpin turn that can be utilized to silence target gene expression via RNAi. Expression of shRNA in cells is generally achieved by delivery of plasmids or through viral or bacterial vectors. Compared to siRNA, shRNA gives benefits in silencing longevity, delivery options and expense. Expressed shRNA is transcribed in cells from a DNA template as a single-stranded RNA (ssRNA) molecule (~50 – 100 bases). Complementary areas spaced by a small 'loop' affect the transcript to fold back on itself forming a 'short hairpin' in a manner analogous to natural microRNA. Recognition and processing via the RNAi machinery the shRNA into the related siRNA.

Knockdown of gene expression at the mRNA level by RNA interference (RNAi) is a common procedure for investigating gene function. For transient knockdown in mammalian cell culture, small interfering RNA (siRNA) is frequently favoured. The advantages of siRNA include commercially accessible RNA oligos that can be transfected into cells for a fast and efficient knockdown. Nonetheless, siRNA becomes less valuable when working with cell types with low transfection efficiency or in experiments that require prolonged gene knockdown. Another common technique for utilizing RNAi is short-hairpin RNA (shRNA), synthetic non-coding RNA which operates the endogenous microRNA machinery to process functional RNAi.

Custom shRNA vectors

Employing our high-intuitive online vector design platform, you can select from over 30 vector backbones (non-viral, viral or transposon) and vast combinations of vector components (promoters, fluorescent and drug-selection markers) for expressing your shRNA. Our shRNA databases enable you to easily select shRNAs against your GOI, without the necessity to design them yourself. We also give shRNA sensor vectors for testing the knockdown efficiency of the shRNA of interest.

Popular shRNA vectors

Seattle Genova offers a panel of popular shRNA vectors that express either scramble shRNA or shRNAs targeting popular genes acceptable for being used as control vectors in several biological applications. 

shRNA virus

Seattle Genova delivers premium quality virus packaging services for lentivirus, AAV and adenovirus in a variety of scales for delivering shRNA into difficult-to-transfect cells. Our proprietary technologies and reagents have vastly improved virus packaging protocols in terms of titer, purity, viability and consistency. Our packaging protocols are also optimized for the viral vector systems utilized in our vector construction services. As a result, we have a growing base of highly convinced customers who come back to us again and again for their cloning and virus packaging requirements.

shRNA databases

Seattle Genova’s online shRNA vector design tool features optimized shRNA databases for common variety, facilitating you to design shRNA vectors with high knockdown efficiency for your target genes. For designing shRNAs, we assign rules like those used by the RNAi consortium. All scores are ≥0, with a mean at ~5, standard deviation at ~5, and 95% of scores ≤15. An shRNA with a knockdown score of about 15 is evaluated to have the best knockdown performance and clonability, while an shRNA with a knockdown score of 0 has the worst knockdown performance or is difficult to be cloned.

Pooled shRNA libraries

Pooled shRNA libraries can fulfil as powerful and cost-efficient tools for conducting large-scale loss-of-function screens for genes comprised in disease pathways, cell responses to drug treatment, developmental processes, gene regulation, etc. We can provide your library as E. coli stock, plasmid DNA pool, or packaged virus, depending on your requirements. Our custom libraries are fully assessed by next-generation sequencing so that you know really what you get.

Workflow for generating stable cell lines 

Transfection - Transfect host cells with recombinant plasmids encoding a protein of concern.

Selection of Pool of Transfected Cells - Select cells that are stable and generate a protein of interest.

Clonal Screening and Selection - Screen and select clones for great expression of the protein of interest.

Cell Line Characterization - Validate and distinguish each clone for stability and productivity.

Expansion and Downstream Evaluation - Expand clones and conduct downstream bioprocesses. Cell banking is also performed.

Deliverable

●Pooled Cells

●3 Single Clones