Site Directed Mutagenesis (SDM) Mouse Deletion

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been greatly used for studies on embryonic development and cell differentiation.iPSCs provide a stable source for either self-renewal or differentiation into suitable cells when cultured in a particular environment. Pluripotent cell culture was originally started by deriving cells from inner cell mass (ICM) from pre-implanted blastocysts, these were called embryonic stem cells. These cells after isolation can be grown on traditional extracellular matrices (like mouse embryonic fibroblasts, MEFs) or feeder-free culture systems. DMEM/F12 has been the most commonly used basal media in the culture of pluripotent cells. These cells are cultured at normal atmospheric oxygen levels, 21%, however, some studies have proposed that 4% oxygen tension may be better for hESC growth. Higher D-glucose concentration (4.2g/l) and osmolarity (320mOsm) that mimics the natural environment of embryonic tissue are optimal for the growth of hESCs. Supplements like N2 and/or B-27, in the presence of growth factors like bFGF, have been shown to increase pluripotency of these cells. bFGF, FGF2 and other ligands of receptor tyrosine kinases like IGF are also required or maintain self-renewal ability of these cells. TGF𝛃1, by its activation of SMAD2/3 signalling, also represses differentiation of iPSCs. Other compounds like ROCK inhibitors reduce blebbing and apoptosis in these cells to maintain their clonogenicity. However, an inhibitor for LIF (leukaemia inhibitory factor, which is one of the pluripotent genes) has an opposing effect. Therefore, it is important to understand the culture conditions and media composition that affect downstream signalling in hESCs or iPSCs that may lead to their differentiation.

Get tips on using FITC Mouse Anti-Ki-67 Set to perform Flow cytometry Anti-bodies Human - Ki-67

Get tips on using PE Mouse anti-Human B7-H4 to perform Flow cytometry Anti-bodies Human - B7-H4

Get tips on using APC Mouse Anti-Human B7-H4 to perform Flow cytometry Anti-bodies Human - B7-H4

Get tips on using APC anti-human/mouse CD49f Antibody to perform Flow cytometry Anti-bodies Human - CD49f/ITGA6

Get tips on using SQSTM1/p62 (D5L7G) Mouse mAb #88588 to perform Autophagy assay cell type - MDA-MB-231

Isolating RNA from tissues and paraffin-embedded tissue samples can be challenging due to cross-linking of biomolecules and fragmented nucleic acids. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in presence of RNAse inhibitors. The homogenization process should be carried out on dry ice to maintain the integrity of RNA.

Get tips on using Mouse Chitinase 3-like 1 Quantikine ELISA Kit to perform ELISA Mouse - Chitinase-3-Like Protein-1 (CHI3L1) or YKL-40

Get tips on using Monoclonal Mouse Anti-Human Cytokeratin (Concentrate) Clone AE1/AE3 to perform Immunohistochemistry Mouse - Cytokeratin

Get tips on using Purified Rat Anti-Mouse CD24 Clone M1/69 (RUO) to perform Immunohistochemistry Mouse - CD24