Abstract
Embryonic stem (ES) cells are an important source of stem cells in tissue engineering and regenerative medicine because of their high self-renewal capacities and differentiation potentials. However, the detailed molecular mechanisms controlling the differentiation and renewal programs in ES cells remained unclear. One of the difficulties in understanding these mechanisms substantially results from the low efficacies of gene manipulation by delivering exogenous gene expression or knockdown of endogenous gene expression with small interfering RNA (siRNA) in ES cells. Here we describe an optimized protocol for efficiently transfecting mouse ES cells by Effectene, a liposome-based method. The high transfection efficiency in mouse ES cells is demonstrated in this chapter by (1) achieving a percentage of enhanced green fluorescence protein (EGFP) expression in >98% embryoid bodies after introducing plasmids encoding the protein; (2) decreased SOX-2 and Oct-3/4 expression and subsequent morphological evidences of cell differentiation after introducing siRNA expression for suppressing SOX-2 and Oct-3/4, which are known to be essential for maintenance of stem cell properties in mouse ES cells; and (3) overexpression or attenuated expression of 14-3-3σ to regulate cell proliferation of mouse ES cells.
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