Chromosome Engineering in Mouse Embryonic Stem Cells: Addition and Elimination of Targeted Chromosomes

Abstract

Human pluripotential stem cells, including both embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), possess self-renewing potency or the ability to differentiate into virtually any type of somatic cell. These features make them particularly advantageous as sources from which to generate specific types of human tissue cells in vitro for use in drug development and regenerative medicine. In most cases, however, human pluripotential cell lines and especially human ES cells can only be used in cell-based applications because of ethical issues. Animal models are therefore sought as an alternative to using human cells. An increasingly popular non-human primate model is the common marmoset (Callithrix jacchus). The recent successful creation of lentivirus-mediated transgenic marmosets provides a new animal model for human disease offering the powerful advantage of a close genetic relationship with humans (Sasaki et al., 2009), though this technique is not yet sufficiently developed for common use, and the numbers of monkeys available for experiments are limited. Mouse ES cells, meanwhile, still have great value as a research tool, even after the development of human pluripotent cells, as they can be used to create chimeric mice, achieve germline transmission, and generate normal offspring. The available genetic engineering technologies often employ an embryonic manipulation approach in mice, using mouse ES cells to examine the gain-of-function or loss-of-function effects associated with certain chromosomal regions in vivo. Recently, we have developed chromosome elimination cassettes (CEC) using a Cre-inverted loxP system that was first used in mouse ES cells. In this system, transient cre expression can initiate immediate chromosomal loss over the course of a few cell cycles in the recombinant cells. This technology was developed to clarify chromosomal function through observing loss-offunction at the chromosomal level. In mammalian cells, chromosome composition and gene dosage are kept stable because large chromosome-wide deletions are usually fatal. Accordingly, we first applied the Cre-inverted loxP system to tetraploid cells composed of mouse ES cells fused with mouse somatic cells to generate conditions conducive to largescale chromosomal imbalance. The CEC-tagged chromosomes could be targets of Credependent chromosome elimination. In addition, we have demonstrated that the Creinverted loxP system enhances cohesion between a loxP site and an adjacent inverted loxP,