RESUMEN
Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line.
Asunto(s)
Células Madre Embrionarias/metabolismo , Marcación de Gen/métodos , Ingeniería Genética/métodos , Hipoxantina Fosforribosiltransferasa/genética , Recombinasas/metabolismo , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Recombinación Genética , TransfecciónRESUMEN
Primary mouse brain cells were cultured with HPRT (hypoxanthine phosphoribosyl transferase)-deficient ES (embryonic stem) cells to see if the ES cells could provide cues sufficient to reprogram a pluripotential state. After 5 days of coculture, HPRT-deficient ES cells were killed by selection in HAT (hypoxanthine, aminopterin, thymidine) medium. We observed islands of HAT-resistant ES-like cells surrounded by differentiated cells. Cell lines generated from three such "islands" proved to be spontaneous, pluripotential ES-neural hybrids, and gave rise to a chimera following blastocyst injection. Re-expression of the ES-specific gene Foxd3 from somatic-derived chromosomes suggested that the somatic nucleus had been reprogrammed. Our results raise the intriguing possibility that ASCs shown to contribute to multiple tissues in blastocyst-injection studies may not contribute as a result of pluripotency. Instead contributions may arise from spontaneous fusion events in which phenotype is determined by either cytoplasmic dominance, nuclear reprogramming, or both.