siRNA / miRNA gene silencing Mouse Neuro 2a

Get tips on using pMSCV-LTR-dCas9-VP64-BFP to perform CRISPR Mouse - Activation Neuro-2a Sim1

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Mouse - Deletion Neuro 2a TET2

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Mouse - Deletion Neuro 2a TSC1

Get tips on using pX330-U6-Chimeric_BB-CBh-hSpCas9 to perform CRISPR Mouse - Deletion Neuro 2a KATNAL2

Get tips on using Silencer® Select- Gdf10 siRNA to perform siRNA / miRNA gene silencing Mouse - 3T3-L1 BMP-3b/GDF10

Get tips on using MISSION® esiRNA_esiRNA targeting mouse Lrp5 (esiRNA1) to perform siRNA / miRNA gene silencing Mouse - MLO‐Y4 Lrp5

Get tips on using MISSION® esiRNA_esiRNA targeting mouse Lrp6 (esiRNA1) to perform siRNA / miRNA gene silencing Mouse - MLO‐Y4 Lrp6

Get tips on using Silencer® Select Negative Control No 1 siRNA to perform siRNA / miRNA gene silencing Mouse - siRNA negative control polymer / lipid

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 SMARTpool: ON-TARGETplus Hipk2 siRNA to perform siRNA / miRNA gene silencing Mouse - Glomerular mesangial cells HIPK2 Polymer / Lipid delivery