Highly stable maintenance of a mouse artificial chromosome in human cells and mice.

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis.

Prognostic significance of sirtuin 2 protein nuclear localization in glioma: an immunohistochemical study.

The sirtuin 2 (SIRT2) protein is a member of the sirtuin family and homologous to Sir2 (silent information regulator 2) of Saccharomyces cerevisiae. To assess the pathobiological significance of SIRT2 protein expression and/or subcellular localization in human glioma, we examined SIRT2 protein expression in human gliomas using a polyclonal anti-SIRT2 antibody and immunohistochemistry. In this study, samples from 23 patients with glioblastoma (GB, grade IV), 8 patients with diffuse astrocytoma (DA, grade II) and 5 healthy individuals were examined. We established a SIRT2 labeling index (SIRT2-LI) that represents the percentage of cells with SIRT2 localized to the nucleus. The mean SIRT2-LI was 65.8±18.6 in GB samples, 41.2±22.8 in DA samples, and 28.6±12.3 in normal control samples. The SIRT2-LI of GB samples was significantly higher than that of normal control samples (P<0.01, Mann-Whitney's U-test) and that of DA samples (P<0.05). Moreover, the SIRT2-LI was positively correlated with malignant progression. Specifically, samples from patients with GB were divided into two groups, low SIRT2-LI (<60%) and high SIRT2-LI (≥60%), and the patients with low SIRT2-LI samples survived significantly longer than patients with high SIRT2-LI samples (P<0.05, Kaplan-Meier method and log-rank test). In conclusion, SIRT2-LI was indicative of glioma malignancy, and it may be predictive of GB patient survival.

Integration-free iPS cells engineered using human artificial chromosome vectors.

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into 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 cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost 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, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.

Integration-free and stable expression of FVIII using a human artificial chromosome.

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.