No abstract

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.

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.

No abstract

We induced apoptosis in HL-60 human promyelocytic leukemia cells by ultraviolet irradiation. Using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and biochemical methods, we studied whether initial deoxyribonucleic acid (DNA) fragmentation could be identified by morphological means in the apoptotic cells. The cell death detection assay revealed oligonucleosomal increase 1.5 h after irradiation, at which time DNA ladder structures appeared on electrophoresis. No smear patterns on electrophoresis and no increase in trypan blue-positive cells denied the probability that this cell model induces necrosis. Light-microscopically, coarse aggregation and nuclear margination of heterochromatin appeared 1.5 h after, then cells with fragmented nuclei increased, and most cells showed apoptotic appearances 4 h after: the findings were confirmed by electron microscopy. These apoptotic cells showed obvious TUNEL signals in nuclei at both lightand electron-microscopic levels. TUNEL signals in heterochromatin of nuclei were faint and diffuse at lh in some normal-looking cells, which lacked DNA ladder pattern. The obtained results indicated the non--random and diffuse cleavage of DNA at the initial stage of apoptosis and validity of TUNEL in identifying apoptotic cells. TUNEL could detect faint changes that appeared earlier when microscopy proved no evidence of apoptosis.Apoptosis is a form of cell suicide (l, 4, 5, ll, l3, l4, l9, 20, 22, 24-26), currently identified by a sequence of morphological and biochemical changes in physiologically dying cells. Morphological changes are distinctive, strikingly illustrating the active nature of the process: cell shrinkage, loss of normal contacts and condensation of nuclear chromatin. The apoptotic cells with nuclear Abbreviati0n.s".' DAB, diaminobenzidine; DNA, deoxyribonucleic acid; dUTP, deoxyuridine 5'-diphosphate; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunoadsorbent assay; RNase, ribonuclease; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP-biotin nick end-labeling; UV, ultraviolet fragmentation subsequently break down into multiple apoptotic bodies, finally phagocytosed rapidly by adjacent cells or macrophages (l4, 27). Biochemically, deoxyribonucleic acid (DNA) isolated from apoptotic cells show a characteristic 'ladder' structure on agarose gel electrophoresis (25).As a useful method to identify apoptotic cells in tissue sections, Gavrieli er al. (8) and others (2, 23) described in siru. end-labeling, known also as terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL). Many researchers have applied this end-labeling procedure in detecting apoptotic cells (2, 7-lO, 12, l5, 18, 23). However, the labeling method has such disadvantages as DNA cleavages are randomly recognized also in necrotic cells (l2). In addition,