Uniform, integration-free iPS cells with a safeguard system using human artificial chromosome vectors

ABSTRACT: Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vector, e.g., episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown construct (see Kazuki et al 2011). The iHAC1 partially reprogrammed MEFs, and the iHAC2 efficiently reprogrammed MEFs. Global gene expression showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. We established iHAC-free iPS cells by isolating cells that spontaneously lost the iHAC2. Analyses of pluripotent markers, teratomas, and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex Virus Thymidine Kinase were eliminated by Ganciclovir treatment; therefore, the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a safeguard system. Total RNA from mouse embryonic fibroblasts, two control ES cells (TT2 and B6ES), retrovirus vector-derived iPS cells (20D17), and twelve HAC-derived iPS clones were hybridized to the Agilent Whole Mouse Genome microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Masaharu Hiratsuka 

PROVIDER: E-GEOD-29441 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress