Immunohistochemistry Wilms Tumor 1 (WT1) Rabbit Mouse

Get tips on using siGENOME Mouse Pard3 (93742) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - MS1 Pard3

Get tips on using IntestiCult™ Organoid Growth Medium (Mouse) to perform 3D Cell Culture Media Mouse gastric cancer organoids

Get tips on using IntestiCult™ Organoid Growth Medium (Mouse) to perform 3D Cell Culture Media Mouse small intestinal organoids

Get tips on using PE anti-mouse/rat CD29 Antibody to perform Flow cytometry Anti-bodies Mouse - CD29/β1-Integrin

Get tips on using siGENOME Mouse Sod2 (20656) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - RGC-5 Sod2

Get tips on using ScriptSeq Complete Kit (Human/Mouse/Rat) to perform RNA sequencing Mouse - Bone marrow-derived macrophages (BMDMs)

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

Get tips on using Accell Mouse Nrep (27528) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - 3T3-L1 P311/Nrep

Get tips on using IntestiCult™ Organoid Growth Medium (Mouse) to perform Stem cell culture media Mouse intestinal stem cells/organoids

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.