Figure 1

A representation of the procedures used to produce autologous stem cells with a corrected genetic defect for the purpose of cell-based gene therapy in adult animals, based on the experiments reported by Rideout et al. [29]. First (top), biopsy samples are obtained from a mutant animal. These are used to establish primary cultures of somatic cells, which will provide donor nuclei. Donor oocytes, arrested at the metaphase II stage of meiosis, are enucleated, and a somatic-cell-derived donor nucleus is transferred into an enucleated oocyte in a procedure known as nuclear transfer. The resulting nuclear-transfer (NT) oocytes are activated and embryogenesis initiates. NT embryos are allowed to develop in vitro up to the blastocyst stage, the stage at which mammalian embryos normally implant into the uterus. For embryonic development to continue, the blastocysts must be reintroduced into the uterus of a (surrogate) female, where they will undergo embryogenesis and ultimately produce cloned mutant offspring. Alternatively, pluripotent NT ES cell lines can be derived from the NT blastocysts. NT ES cells bear all the hallmarks of standard ES cell lines, in that they exhibit broad (pluripotent) developmental potential, their genome can be manipulated in vitro by routine gene targeting and other transgenic approaches, and they can be differentiated in vitro when grown under appropriate conditions. Gene targeting can be used to repair specific genetic defects in the mutant NT ES cells. The corrected NT ES cells can subsequently be introduced into tetraploid blastocysts to generate chimeras that, if implanted into the uterus of a surrogate female and allowed to undergo embryogenesis, can develop into cloned normal offspring. Alternatively, corrected NT ES cells can be differentiated in vitro to obtain lineage-specific stem cells, in this case by growth of embryoid bodies (EBs) followed by differentiation to yield hematopoietic stem cells. Finally, the genetically corrected, autologous cells can be used for cell therapy of the mutant animals.