protein-expression-and-purification-mammalian-cells-hek-293-mt-pa

Get tips on using pET24b-prochymosin to perform Protein Expression Prokaryotic cells - E. coli calf prochymosin

Get tips on using pET28a/VDH to perform Protein Expression Prokaryotic cells - E. coli vanillin dehydrogenase

Get tips on using pET28b-MPMVGag to perform Protein Expression Prokaryotic cells - E. coli (MPMV) Pr78Gag

Get tips on using pTrcHis-TOPO to perform Protein Expression Prokaryotic cells - E. coli Murine BCO2

Get tips on using pG-S1D to perform Protein Expression Prokaryotic cells - E. coli rGST-S1D

Get tips on using pG-COE to perform Protein Expression Prokaryotic cells - E. coli rGST-COE

Get tips on using pET28a+-BL1 to perform Protein Expression Prokaryotic cells - E. coli DsbA BL1

Get tips on using pSB 130 to perform Protein Expression Prokaryotic cells - A. tumefaciens ELP-intein

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 have 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 efficacy of transduction and shRNA on its target site.

Get tips on using pFlpBtM-II-eGFP to perform Protein Expression Eukaryotic cells - S. frugiperda EGFP