A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression

Immortalization of Cells from Brains Derived from a Strain (MSM/MSfB6C3F1) of Wild Mouse

Escape from cellular aging is the rate-limiting step of multistep carcinogenesis. While normal human cells invariably undergo cellular aging and almost never spontaneously immortalize, cells derived from rodents such as mice are relatively easily immortalized. In this experiment, we studied the immortalization patterns of cells obtained from brain tissues of an inbred strain (MSM/MSfB6C3F1) derived from wild mice. We established 12 cell strains derived from 12 mouse brains in order to investigate whether these cells show cellular aging in the same fashion as human cells or whether these cells are immortalized as easily as rodent cells reported previously. As a result, all cell strains were immortalized up to about 200 days in culture. One strain immortalized very early, in the first 50 days, four strains immortalized in the last 200 days, and the other seven strains became immortal between 150 and 200 days in culture. All immortalized cell strains showed varying amounts of chromosome abnormalities, numerically and structurally, but no specific changes related to immortalization were detected. Before immortalization, three types of cells, glial-like, polygonal flat-thin, and fibroblast-like cells, were observed in culture, but after immortalization most of the cultures became fibroblastic. From these results, we concluded that fibroblast-like cells derived from brains of these mice immortalized in like fashion to fibroblasts of other inbred mice.</p

Regulation of functional KCNQ1OT1 lncRNA by β-catenin.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes through epigenetic mechanisms. We previously reported that KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster. Furthermore, aberration of KCNQ1OT1 transcription was observed with a high frequency in colorectal cancers. However, the molecular mechanism of the transcriptional regulation and the functional role of KCNQ1OT1 in colorectal cancer remain unclear. Here, we show that the KCNQ1OT1 transcriptional level was significantly increased in human colorectal cancer cells in which β-catenin was excessively accumulated in the nucleus. Additionally, overexpression of β-catenin resulted in an increase in KCNQ1OT1 lncRNA-coated territory. On the other hand, knockdown of β-catenin resulted in significant decrease of KCNQ1OT1 lncRNA-coated territory and an increase in the mRNA expression of the SLC22A18 and PHLDA2 genes that are regulated by KCNQ1OT1. We showed that β-catenin can promote KCNQ1OT1 transcription through direct binding to the KCNQ1OT1 promoter. Our evidence indicates that β-catenin signaling may contribute to development of colorectal cancer by functioning as a novel lncRNA regulatory factor via direct targeting of KCNQ1OT1

Dynamics of epigenetic regulation at the single-cell level

Chromatin regulators play a major role in establishing and maintaining gene expression states. Yet how they control gene expression in single cells, quantitatively and over time, remains unclear. We used time-lapse microscopy to analyze the dynamic effects of four silencers associated with diverse modifications: DNA methylation, histone deacetylation, and histone methylation. For all regulators, silencing and reactivation occurred in all-or-none events, enabling the regulators to modulate the fraction of cells silenced rather than the amount of gene expression. These dynamics could be described by a three-state model involving stochastic transitions between active, reversibly silent, and irreversibly silent states. Through their individual transition rates, these regulators operate over different time scales and generate distinct types of epigenetic memory. Our results provide a framework for understanding and engineering mammalian chromatin regulation and epigenetic memory

Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements

黒色腫細胞において、miR-19bはPITX1 mRNAを標的としてhTERT mRNAの発現を制御する

Purpose: This article sheds light onto the increasing problem of product returns, which is exacerbated by growing e-commerce. Many retailers and academics are oblivious to the nature and scale of this challenge. Interdisciplinary research is needed to develop supporting theory, and cross-functional teams are required to implement measures addressing economic, ecological and social sustainability issues. Design/methodology/approach: The initial project adopted a multi-case study approach, whereby returns processes were mapped, vulnerabilities identified and a returns cost calculator was developed. Findings: Product returns processes are usually complicated, prone to internal and external fraud, inefficient and lack sustainability. They can generate considerable losses to the business, especially as returns data are often not systematically collected, monitored or reported to senior management. There are important implications for strategic and operational management, namely the need to develop a concept for Lean returns systems. Originality/value: Product returns are a unique and understudied but growing field in academic research, with only few publications over the last two decades. Yet the phenomenon is causing increasing problems in business and society. Robust solutions could achieve great financial and non-financial impacts.</p