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Background and Purpose: The expression of voltage-dependent K(+) channels (K(v) ) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF-1) and its inducer geranylgeranylacetone (GGA) could affect the expression of K(v) 1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97).Experimental Approach: Transfected mouse atrial cardiomyocytes (HL-1 cells) and COS7 cells were subjected to luciferase reporter gene assay and whole-cell patch clamp. Protein and mRNA extracts were subjected to Western blot and quantitative real-time polymerase chain reaction.Key Results: Heat shock of HL-1 cells induced expression of Hsp70, HSF-1, SAP97 and K(v) 1.5 proteins. These effects were reproduced by wild-type HSF-1. Both heat shock and expression of HSF-1, but not the R71G mutant, increased the SAP97 mRNA level. Small interfering RNA (siRNA) against SAP97 abolished HSF-1-induced increase of K(v) 1.5 and SAP97 proteins. A luciferase reporter gene assay revealed that the SAP97 promoter region (from -919 to -740) that contains heat shock elements (HSEs) was required for this induction. Suppression of SIRT1 function either by nicotinamide or siRNA decreased the level of SAP97 mRNA. SIRT1 activation by resveratrol had opposing effects. A treatment of the cells with GGA increased the level of SAP97 mRNA, K(v) 1.5 proteins and I(Kur) current, which could be modified with either resveratrol or nicotinamide.Conclusions and Implications: HSF-1 induced transcription of SAP97 through SIRT1-dependent interaction with HSEs; the increase in SAP97 resulted in stabilization of K(v)1.5 channels. These effects were mimicked by GGA. [ABSTRACT FROM AUTHOR]

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis. [ABSTRACT FROM AUTHOR]

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53−/− mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.Gene Therapy (2008) 15, 617–624; doi:10.1038/sj.gt.3303091; published online 28 February 2008 [ABSTRACT FROM AUTHOR]

One of the obstacles in studying human genomic imprinting is distinguishing the parental origin of alleles in diploid cells. To solve this problem, we have constructed a library of mouse A9 hybrids in which individual clones contain a single human chromosome of known parental origin. Here we extend this in vitro system to the analysis of the role of histone acetylation in the allelic expression of human imprinted genes. The levels of histone H4 acetylation of the imprinted human LIT1, H19, and SNRPN genes were examined by a chromatin immunoprecipitation (ChIP) assay in mouse A9 hybrids with a single human chromosome of known parental origin. We demonstrated that H4 histones associated with the actively expressed alleles of imprinted LIT1, H19, and SNRPN genes were highly acetylated, whereas they were hypoacetylated in the silent alleles. Furthermore, treatment of A9 hybrids with trichostatin A (TSA), an inhibitor of histone deacetylase, resulted in transcriptional reactivation of the silent alleles for LIT1 and SNRPN, suggesting that histone deacetylation is one of the key regulatory mechanisms in genomic imprinting. These results indicate that our monochromosomal hybrid system is a new technology for analyzing histone modifications between parental alleles in human imprinted genes. [ABSTRACT FROM AUTHOR]