Protein expression and purification Mammalian cells HeLa

Get tips on using pMCSG7-PP-CX to perform Protein Expression Prokaryotic cells - E. coli PP-CX

Get tips on using pTRAc/pRIC 3.0 to perform Protein Expression Prokaryotic cells - A. tumefaciens BFDV cp

Get tips on using pUC19-Pgpd-xyn2-Tgpd to perform Protein Expression Eukaryotic cells - T. reesei xyn2

Get tips on using OmpA-TRAIL/pET-22b to perform Protein Expression Prokaryotic cells - E. coli TRAIL

Get tips on using pMmEG(TA)-rhBMP-4 to perform Protein Expression Eukaryotic cells - CHO BMP-4

Get tips on using IGEPAL® CA-630 to perform Protein isolation Mammalian cells - Human lung fibroblasts

Get tips on using pIEX-5-6His-mMBP-UMODpXR to perform Protein Expression Eukaryotic cells - HEK293 mMBP

Get tips on using p1.2-Hygro-FSH-B-chain to perform Protein Expression Eukaryotic cells - CHO FSH

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.

Get tips on using QuantiPro™ BCA Assay Kit to perform Protein quantification Mammalian cells - HEK 293