The use of gating technology in bioengineering insulin-secreting cells from embryonic stem cells.

Embryonic stem cells display the ability to differentiate in vitro into a variety of cell types. This process is induced by embryoid body formation, addition of several soluble growth factors to the culture medium and other strategies. However, none of the used factors is capable to drive differentiation to only one specific celltype. The use of gating technology has allowed to partially overcome this problem. The rational behind this technique is based on the transfection of stem cells with a transgene carrying expression cassettes for a cell type specific promoter, regulating expression ofa selectable marker to select one cell lineage from other cell lineages.Using this system, we have obtained insulin-secreting cells by transfecting mouse embryonic stem cells with a DNA construct providing resistance to neomycin under the control of the regulatory regions of the human insulin gene. Furthermore, gating technology has been successfully used to isolate other cell types such as cardiomyocytes and neural precursors from undifferentiated embryonic stem cells. This review focuses on the possibilities offered by this technology in embryonic stem cell bioengineering, mainly to obtain insulin-secreting cells. Advantages and considerations of this selection system will be also discussed.

Ligand-inducible transgene regulation for gene therapy.

A synthetic ligand regulable system for gene transfer and expression has been developed in our laboratory based on mechanistic studies of steriod hormone receptor and transcriptional regulation. This gene switch system possesses most of the important features that are required for application of the system in biological research and clinical gene therapy in the future. As the primary ligand tested in this system, mifepristone can effectively turn on the regulatory circuit at doses much lower than those used in the clinic. By modification of the chimeric regulator and its feedback regulatory mode, this system has been optimized to produce very low basal activity with high inducibility in the presence of mifepristone. Also, improvements in regulator composition have been made to minimize immunogenicity and make the system more amenable to human gene therapy. Moreover, incorporation of this gene switch system into the HC-Ad vector system has further enhanced the efficiency of gene transfer and the long-term inducible expression of transgenes. However, for each application within a different biological system, the gene switch needs to be optimized to achieve appropriate inductions. In particular, the method used to deliver the transgenes and adjustment of ligand dosage are critical for in vivo gene expression.