Sigma-Aldrich Product Name Mbd3KO, 11041301 biological source mouse embryo growth mode Adherent

Mouse embryonic stem cell; Mbd3 knock-out

Mbd3KO stem cells have been genetically modified to produce a null phenotype for Mbd3, a critical determinant for lineage commitment in embryonic stem cells. This lack of Mbd3 (Mbd3 exons 2-7 have been replaced by an IRES-βgeo-pA sequence) allows them to be maintained in the absence of any exogenous factors and they fail to commit to developmental lineages. Upon re-introduction of the Mbd3 gene the cells are able to differentiate into identifiable bone, muscle, skin, fat and neuronal tissue. Mbd3 null embryonic stem cells can be used to study LIF-independent self-renewal to develop human stem cell lines and new cell culture media. They can also be used to develop specific stem cell lines for high throughput screening of therapeutic compounds. Finally they can be used to develop transgenics for the study of specific diseases or for the development of specific therapeutic stem cell lines for replacement therapies to treat certain diseases.

GMEM (G5154) + 2 mM L-Glutamine (G7513) + 10% FBS / FCS (F2442) + 0.1 mM 2-Mercaptoethanol (M6250) + 1000 Units/ml LIF. Use of LIF is optional, but its addition reduces differentiation.

Plastic ware is pre-coated with gelatine as follows: porcine gelatine (Sigma product number G1890) is dissolved in sterile water (0.5 g/500ml) at 50°C. The 0.1% solution is sterilized by filtration (0.22 μm). Add 0.1% gelatine to plastic ware to cover bottom, and incubate for 20 minutes at room temperature . Remove gelatine, wash with PBS once and replace with appropriate culture medium. The flask/dish must not be allowed to dry out. An ampoule of Mbd3KO cells is thawed in 37 °C water bath and the contents quickly transferred to a 15 ml centrifuge tube. Medium is added drop wise to 5 ml. Cells are centrifuged at 150 x g for 5 minutes. Cells are resuspended in 5 ml of medium. Cells should be plated at 4-5 x 10⁴ cells/cm² using gelatine coated flasks. Cells must be subcultured every 2-3 days using 0.25% trypsin or trypsin/EDTA. Colonies must not be allowed to touch each other as overgrowth will result in differentiation. Cultures must be incubated in a humidified 5% CO₂/95% air incubator at 37 °C.

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