One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes and studying their function, and are now being initiated in human ES (hES) cells. This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of hES cells and their applications. Specifically, two detailed protocols that can be used to generate clones of genetically modified hES cells by transfection will be described, with special emphasis on the important technical details that are required for this purpose.
Key Words: Human ES cells; genetic manipulation; transfection; overexpression; targeted mutagenesis; homologous recombination; knock-down by RNAi.
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