CRISPR Mouse Deletion ES (embryonic stem) cells

Get tips on using Gibco™DMEM, low glucose, pyruvate to perform Stem cell culture media Human bone mesenchymal stem cell (BMSC)

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

Get tips on using Phusion Site-Directed Mutagenesis Kit to perform Site Directed Mutagenesis (SDM) Rat - Deletion H9C2 SRF

Get tips on using SurePrint G3 Mouse Exon 4x180K Microarray Kit (165,984 Exon probes) to perform Microarray Mice - Cochlea Expression array (labelled)

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 Cas9m4-VP64 to perform CRISPR Human - Activation NEUROD1

Get tips on using lentiGuide-Puro to perform CRISPR Human - Repression BIRC5

Get tips on using lentiGuide-Puro to perform CRISPR Human - Repression BIRC5

Get tips on using lentiCRISPR v2 to perform CRISPR Human - Repression BCL6

Get tips on using lentiCRISPR v2 to perform CRISPR Human - Repression PCSK9