Site Directed Mutagenesis (SDM) Mouse Point mutation C2C12

Get tips on using ChIP-IT® Express Chromatin Immunoprecipitation Kits to perform ChIP Mouse - Brain

Get tips on using ChIP-IT® Express Chromatin Immunoprecipitation Kits to perform ChIP Mouse - HT22

Get tips on using ChIP-IT® Express Chromatin Immunoprecipitation Kits to perform ChIP Mouse - NIH3T3

Get tips on using EZ DNA Methylation kit to perform DNA methylation profiling Whole genome profiling - mouse hematopoietic stem cells

Get tips on using pSpCas9(BB)-2A-Puro (PX459) to perform CRISPR Mouse - Deletion ES (embryonic stem) cells Etv2 promoter

Get tips on using Dulbecco’s Modified Eagle’s Medium - high glucose to perform Stem cell culture media Mouse fibroblasts from meninges

Get tips on using Gibco™Ham's F-10 Nutrient Mix to perform Stem cell culture media Mouse myoblasts cells

Get tips on using KAPA RNA HyperPrep Kit with RiboErase (HMR) to perform RNA sequencing Mouse - ESCs (Embryonic Stem Cells)

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.

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.