CRISPR Mouse Deletion NSC34

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.

Get tips on using Purified Mouse Anti-Human MSH-2 Clone G219-1129 (RUO) to perform Immunohistochemistry Human - MSH2

Get tips on using Monoclonal Mouse Anti-Villin (Autostainer Link 48) Clone 1D2 C3 to perform Immunohistochemistry Human - Villin

Get tips on using Monoclonal Mouse Anti-Human Progesterone Receptor (Concentrate) Clone PgR 1294 to perform Immunohistochemistry Human - PR

Get tips on using Monoclonal Mouse Anti-Human CA 125 (Dako Omnis) Clone M11 to perform Immunohistochemistry Human - CA125

Get tips on using Monoclonal Mouse Anti-Human CDX2 (Dako Omnis) Clone DAK-CDX2 to perform Immunohistochemistry Human - CDX2

Get tips on using Monoclonal Anti-Proliferating Cell Nuclear Antigen antibody produced in mouse to perform Western blotting PCNA

Get tips on using Monoclonal Mouse Anti-Villin (Autostainer Link 48) Clone 1D2 C3p to perform DNA Damage Assay U266 -

Get tips on using Purified Mouse Anti-E-Cadherin Clone 36/E-Cadherin (RUO) to perform Immunohistochemistry Human - E-Cadherin

Get tips on using Monoclonal Mouse Anti-Thyroid Transcription Factor (Concentrate) Clone 8G7G3/1 to perform Immunohistochemistry Human - TTF-1