Site Directed Mutagenesis (SDM) Mouse Deletion C2C12

Get tips on using Alexa Fluor® 700 Mouse Anti-Mouse NK1.1 to perform Flow cytometry Anti-bodies Mouse - NK1.1

Get tips on using DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - C2C12

Get tips on using Cell DNA Isolation Kit to perform DNA isolation / purification Cells - Immortalized cell lines C2C12

Get tips on using DNeasy Blood & Tissue Kit to perform DNA isolation / purification Cells - Immortalized cell lines C2C12

Get tips on using CellTiter-Glo® Luminescent Cell Viability Assay to perform Live / Dead assay mammalian cells - C2C12

Get tips on using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit to perform ROS assay cell type - C2C12

Get tips on using Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) to perform Flow cytometry Anti-bodies Mouse - CD16/CD32

Get tips on using siGENOME Mouse Alox12 siRNA to perform siRNA / miRNA gene silencing Mouse - B16-F10 12-Lox/ALOX12

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.

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.