Site Directed Mutagenesis (SDM) Mouse L929

Though DNA quantification is but one small step in the multifaceted DNA sample preparation workflow, it can have large implications on the performance and validity of conclusions drawn from downstream assays. Major challenges include accuracy, precision, reproducibility, and detection of present contamination. Among UV spectrophotometry, fluorescence and real-time PCR based methods, the quantification method should be chosen based on the requirement of the downstream assay.

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

Get tips on using ON-TARGETplus Mouse Cflar (12633) siRNA to perform siRNA / miRNA gene silencing Mouse - 3T3-SA Flip/CFLAR

Get tips on using Purified Rat Anti-Mouse CD31 to perform Immunohistochemistry Mouse - CD31

Get tips on using Biotin Rat Anti-Mouse CD24 to perform Immunohistochemistry Mouse - CD24

Get tips on using Mouse/Rat Neuropilin-1 Antibody to perform Immunohistochemistry Mouse - Nrp1

Get tips on using Mouse VEGF ELISA Kit (ab100751) to perform ELISA Mouse - VEGF

Get tips on using Mouse VEGF Quantikine ELISA Kit to perform ELISA Mouse - VEGF

Get tips on using Mouse Shh-N ELISA Kit to perform ELISA Mouse - ShhN