shRNA gene silencing Human HEK 293T

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 pcDNA3.1-MRP4-his6 to perform Protein Expression Eukaryotic cells - HEK293 human MRP4-his6

Get tips on using TruSeq Small RNA Library Preparation Kit to perform RNA sequencing Human - HEK293T

Get tips on using TruSeq RNA Library Prep Kit v2 to perform RNA sequencing Human - HEK293T

Get tips on using EpiTect MSP Kit to perform DNA methylation profiling Gene specific profiling - HeLa MEG3

Get tips on using EpiTect Bisulfite Kit to perform DNA methylation profiling Gene specific profiling - HepG2 Fibrinogen

Get tips on using Phusion Site-Directed Mutagenesis Kit to perform Site Directed Mutagenesis (SDM) Human - Point mutation HEK293 MDR3

Microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - Hep3B

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - HepG2