Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

<p>Abstract</p> <p>Background</p> <p>Cynomolgus macaques (<it>Macaca fascicularis</it>) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.</p> <p>Results</p> <p>We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.</p> <p>Conclusion</p> <p>BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.</p

Dynamics of cellular immune responses in the acute phase of dengue virus infection.

In this study, we examined the dynamics of cellular immune responses in the acute phase of dengue virus (DENV) infection in a marmoset model. Here, we found that DENV infection in marmosets greatly induced responses of CD4/CD8 central memory T and NKT cells. Interestingly, the strength of the immune response was greater in animals infected with a dengue fever strain than in those infected with a dengue hemorrhagic fever strain of DENV. In contrast, when animals were re-challenged with the same DENV strain used for primary infection, the neutralizing antibody induced appeared to play a critical role in sterilizing inhibition against viral replication, resulting in strong but delayed responses of CD4/CD8 central memory T and NKT cells. The results in this study may help to better understand the dynamics of cellular and humoral immune responses in the control of DENV infection

ニホンザル ニ オケル カルレティキュリン ノ コウゾウ ト ソシキ ハツゲン

京都大学0048新制・課程博士博士(理学)甲第12871号理博第3181号新制||理||1470(附属図書館)UT51-2007-H144京都大学大学院理学研究科生物科学専攻(主査)教授 景山 節, 教授 平井 啓久, 教授 渡邊 邦夫学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDA

Transcriptional dynamics of immortalized human mesenchymal stem cells during transformation

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5(GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC)

Efficient in vivo depletion of CD8(+) T lymphocytes in common marmosets by novel CD8 monoclonal antibody administration.

In order to directly demonstrate the roles of CD8(+) T lymphocytes in non-human primates, in vivo depletion of the CD8(+) T cells by administration of a CD8-specific monoclonal antibody (mAb) is one of the crucial techniques. Recently, the common marmoset (Callithrix jacchus), which is classified as a New World monkey, has been shown useful as an experimental animal model for various human diseases such as multiple sclerosis, Parkinson's disease and a number of infectious diseases. Here we show that an anti-marmoset CD8 mAb 6F10, which we have recently established, efficiently depletes the marmoset CD8(+) T lymphocytes in vivo, i.e., the administration of 6F10 induces drastic and specific reduction in the ratio of the CD8(+) T cell subset for at least three weeks or longer. Our finding will help understand the pivotal role of CD8(+) T cells in vivo in the control of human diseases

Phenotypic characteristics of UE6E7T-3 during long-term <i>in vitro</i> culture.

<p>(<b>A</b>) Growth curve. UE6E7T-3 cells were seeded at 5X10³ cells / ml in POWEREDBY 10. When cells were subconfluently grown, the cells were passaged with trypsin as shown in the text. After counting cell numbers, aliquot of the cultured cells was cultured continuously. (<b>B</b>) Phase-contrast images of UE6E7T-3 at four stages. Scale bar, 300 μm. (<b>C</b>) Flow cytometry. UE6E7T-3 cells (10,000 cells) were plotted. X-axis is fluorescence intensity. Y-axis is number of cell.</p

Phenotypic alterations of UE6E7T-3 during long-term culture.

<p>(<b>A</b>) Changes in chromosomal number at various culture stages. Chromosomes were counted by DAPI staining of 50–80 metaphase spreads at each PDL. Distribution pattern of cells at PDL 62, 92, 118, and 147 were rearranged from raw data from a previous report. (<b>B</b>) Multicolored fluorescence <i>in situ</i> hybridization (mFISH) karyotyping of UE6E7T-3 at three culture stages. Chromosome 13 is circled. (<b>C</b>) Colony formation in soft agar at three culture stages. (<b>D)</b> Graphical representation of the relative colony counts (expressed as percentages) at four culture stages. Colonies ≥300 μm in diameter were counted. Bars represent mean numbers of colonies of triplicates (percent + s.d.). ✰, p < 0.01. (<b>E</b>) Hematoxylin and eosin staining of invasive sarcoma following i.m. injection of U3-DT. Scale bar, 500μm. (Boxed area is magnified in D). (<b>F</b>) Magnification of transformed U3-DT in mouse muscle tissue. Dividing cells (arrow), cells containing two nuclear bodies (arrowhead), and cells containing increased chromatin (asterisk) indicate tumorigenesis. Scale bar, 50 μm.</p