Site Directed Mutagenesis (SDM) Mouse 3T3-L1

Get tips on using Quant-iT™ PicoGreen® dsDNA Assay Kit to perform DNA quantification Mouse - NIH 3T3

Get tips on using CytoSelect™ 24-Well Wound Healing Assay to perform Wound healing assay cell type - mouse NIH 3T3

Get tips on using GeneArt™ CRISPR Nuclease Vector with CD4 Enrichment Kit to perform CRISPR Mouse - Deletion NIH 3T3 G3BP

Get tips on using GeneArt™ CRISPR Nuclease Vector with CD4 Enrichment Kit to perform CRISPR Mouse - Deletion NIH 3T3 FXR

Get tips on using FITC Mouse Anti-Mouse NK-1.1 to perform Flow cytometry Anti-bodies Mouse - NK1.1

Get tips on using APC Mouse Anti-Mouse NK-1.1 to perform Flow cytometry Anti-bodies Mouse - NK1.1

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 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.