Generation of human artificial chromosomes in human embryonic stem and neuronal cells

De novo human artificial chromosomes (HAC) are non-integrating, high capacity vectors which function as stable normal chromosomes, and can incorporate large genes and the surrounding regulatory regions. HAC are composed of euchromatin and heterochromatin, and are models for investigating chromosome organization and gene expression. HAC are generated following transfer of large arrays of core centromeric DNA (alpha satellite DNA) into target cells. We developed a highly efficient delivery protocol based on the Herpes Simplex Virus type 1 (HSV-1) amplicon mediated transduction of cells, which targeted large DNA to the cell nucleus of different cell types. Recently HAC were stably generated at high frequency in human embryonic stem cells (hESc).  Unlike tumour cells, where the input DNA often integrated in the host chromosomes, no integrated DNA was found in hESc. The HAC hES cells retained pluripotency and teratoma formation capabilities and were successfully differentiated into neuronal cells.