Identifying new aggrecanase and producing antibody and gene-targeting mouse

为了研究新的聚合酶，我们做了以下实验，得到了一些数据：1)设计了各种针对ADAMTS的特异性引物，并进行了RT-PCR。我们培养了人软骨肉瘤细胞系OUMS-27(冈山大学医学院-27)，并用白细胞介素1β(IL-1β)刺激。为了定量检测基因的表达，我们采用了实时荧光定量RT-PCR方法。简单地说，提取总RNA，然后进行DNA酶处理以消除污染的基因组DNA。用随机引物和酶逆转录后，以cDNA3为模板进行RT-PCR。2)IL-1β的表达和基因调控不同于体外报道的具有聚集素切割活性的ADAMTS-1、4和-5。我们还研究了其他ADAMTS基因的表达。3)我们在OUMS-27细胞中发现了另一个被IL-1β上调的ADAMTS基因。我们利用该ADAMTS的多肽序列制备了针对该新基因的多克隆抗体。4)我们还开始制作该基因的敲除小鼠。我们筛选了小鼠基因组文库，并鉴定了包括这个新的ADAMTS基因的几个克隆。在与阿普特博士在美国的实验室的合作下，我们开始将我们的克隆移植到ES细胞中。

To investigate new aggrecanase, we have done the following experiments and got some data.1) Various ADAMTS-specific primers were designed and RT-PCR was performed. We cultured human chondrosarcoma cell lines OUMS-27 (Okayama University Medical School-27) and stimulated with interleukine-1 beta (IL-1β). To determine gene expression quantitatively, we employed real-time RT-PCR method. Briefly, total RNA was extracted and then DNAse treatmeat was done to eliminate contaminating genomic DNA. After reverse transcribed with random primers and enzymes, cDNA was served as a template for RT-PCR. GAPDH was used for the internal control.2) The expression and gene regulation by IL-1β was different amonf the ADAMTS-1,4, and -5, which were reported to have aggrecan cleaving property in vitro. We also investigated other ADAMTS gene expressions.3) We identified another up-regulating ADAMTS gene by IL-1β in OUMS-27 cells. We raised polyclonal antibody against this new ADAMTS gene using peptide sequence of this ADAMTS.4) We also started to making knock-out mouse for this gene. We screened mouse genomic library and identified several clones including this new ADAMTS gene. Under the collaboration with Dr. Apte's lab in the USA, we started to put our clones to ES cells.