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

BRCA1 haploinsufficiency promotes chromosomal amplification under Fenton reaction-based carcinogenesis through ferroptosis-resistance.

Germline-mutation in BRCA1 tumor suppressor gene is an established risk for carcinogenesis not only in females but also in males. Deficiency in the repair of DNA double-strand breaks is hypothesized as a responsible mechanism for carcinogenesis. However, supporting data is insufficient both in the mutation spectra of cancers in the patients with BRCA1 germline-mutation and in murine knockout/knock-in models of Brca1 haploinsufficiency. Furthermore, information on the driving force toward carcinogenesis in BRCA1 mutation carriers is lacking. Here we applied Fenton reaction-based renal carcinogenesis to a rat heterozygously knockout model of BRCA1 haploinsufficiency (mutant [MUT] model; L63X/+). Rat MUT model revealed significant promotion of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Array-based comparative genome hybridization of the RCCs identified significant increase in chromosomal amplification, syntenic to those in breast cancers of BRCA1 mutation carriers, including c-Myc, in comparison to those in the wild-type. Subacute-phase analysis of the kidney after repeated Fe-NTA treatment in the MUT model revealed dysregulated iron metabolism with mitochondrial malfunction assessed by expression microarray and electron microscopy, leading to renal tubular proliferation with iron overload. In conclusion, we for the first time demonstrate that biallelic wild-type BRCA1 provides more robust protection for mitochondrial metabolism under iron-catalyzed oxidative stress, preventing the emergence of neoplastic cells with chromosomal amplification. Our results suggest that oxidative stress via excess iron is a major driving force for carcinogenesis in BRCA1 haploinsufficiency, which can be a target for cancer prevention and therapeutics

Environmental Enrichment Increases Radiation-induced Apoptosis Not Spontaneous Apoptosis in Mouse Intestinal Crypt Cells

Abstract. Background: An enriched environment (EE) as an animal housing condition modifies apoptotic cell death and promotes cell proliferation in the central nervous system (CNS). However, few studies have examined the effects of an EE on apoptosis in non-CNS organs. Here we evaluated the effects of an EE on spontaneous and radiation-induced apoptosis of intestinal crypt cells. Materials and Methods: Juvenile (3 weeks after birth) and adult (11 weeks) male B6C3F1 mice were housed in a standard environment or EE for 8 weeks and then whole-body irradiated with 2 Gy X-rays. Apoptosis in the small intestine and colon was analyzed histologically by immunohistochemically using anti-cleaved caspase 3 antibody. Results: The EE used in this study significantly reduced body weight, adipose tissue weight, and serum levels of total-cholesterol, triglyceride, leptin, and insulin. Notably, although EE did not change the spontaneous apoptotic index without irradiation, it significantly increased the index after irradiation in the colonic crypt. Apoptosis therein was enhanced in the region where putative stem/progenitor cells are located. The apoptotic index in the small intestinal crypt showed similar patterns. Conclusion: EE enhances susceptibility to radiation-induced apoptosis of stem/progenitor cells in both small intestine and colon without affecting spontaneous apoptosis

Synthetic Photochemistry. XLIII. Chromium (II) Chloride-Mediated Reductive Condensations of (3S)- and (3R)-1-Iriden-7-als with (3S)-7-Chloro-1-iridenes. ^1H NMR Spectral Differentiation of the Stereochemistry

The chromium (II) chloride-mediated condensations of the several (3S)-and (3R)-1-iriden-7-als with (3S)-7-chloro-1-iridenes, were carried out. Due to a large steric hindrance from the C_3-side chain of the iridenes, stereochemistry of resultant condensates always possessed cis-geometry for the C-11 methyl and the C-14 C_3-substituents. The ^1H NMR chemical shift comparisons established the orientation of the hydroxyl groups on the C-1