follistatin-related protein : analysis of arthritis induced in its transgenic mice

卵泡抑素相关蛋白：转基因小鼠诱导关节炎的分析

• 批准号: 11557038
• 负责人: OZAKI Shoichi
• 金额: $ 8.51万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557038/
• 关键词: FRP; transgenic mice; transgene; type II collagen; PCR; FLAG epitope; DBA; 1 mouse; II型コラーゲンプロモーター; FLAGエピトープタグ; 1マウス; Iマウス

To produce the transgenic mice, which can overexpress follistatin-related protein (FRP) specifically in the articular cartilage, the transgenes named Col2a1-mFRP and Col2a1-FLAG-mFRP were constructed. In both transgenes, mouse FRP (mFRP) cDNA was arranged downstream to the promoter of type II collagen. In Col2a1-FLAG-mFRP, the 5' nucleotide sequence of mFRP cDNA was modified to generate the protein bearing a FLAG epitope tag in the N terminus. To make sure that the transgenes could be transcribed under the regulation of type II collagen promoters, Col2a1-FLAG-mFRP was transferred to a mouse teratoma cell line ATDC5 in the developing stage to chondrocytes, and the transcribed mRNA was estimated. FLAG-mFRP mRNA was certainly detected by RT-PCR with its gene-specific primers. In this experiment producing transgenic mice, Col2a1-FLAG-mFRP of the two transgenes was adopted for the easy detection of the gene products by FLAG epitope tags. Col2a1-FLAG-mFRP was injected into embryos of DBA/1 (Crj) mice, and morphologically intact embryos were transferred to the uterine tubes of ICR mice. 310 embryos out of 344 transgene-injected ones were transplanted to eleven mice, and two mice became pregnant and bore four F0 offspring. RT-PCR analysis of genomic DNA with FLAG-mFRP-specific primers detected the transgenes in only one F0 mouse. This transgene-positive F0 mouse had almost the same phenotype as the wild type mouse. We are now producing F1 mice by mating this F0 mouse with wild DBA/1 (Crj) mice, and analyzing the transgenes in the born 53 F1 mice. As a control study, we performed arthritis-induction experiments in wild type mice with monoclonal antibodies to collagen (Arthritogenic mAb Cocktail, IBL), and succeeded in causing arthritis in all the tested mice. From now on, we will continue the transgene injection to the embryos in order to get more candidate strains of transgenic mice in this study. This project is now under way.

为了获得在关节软骨中高表达卵泡抑素相关蛋白(FRP)的转基因小鼠，构建了转基因小鼠，命名为Col2a1-MFRP和Col2a1-FLAG-MFRP。在两种转基因中，小鼠FRP(MFRP)基因均位于II型胶原启动子下游。在Col2a1-FLAG-MFRP中，对MFRP的5‘核苷酸序列进行了修饰，在N端产生了带有标志表位标签的蛋白质。为了确保转基因能够在II型胶原启动子的调控下转录，将Col2a1-FLAG-MFRP转移到发育阶段的小鼠畸胎瘤细胞系ATDC5中，并检测其转录水平。用FLAG-MFRP基因特异性引物RT-PCR检测到FLAG-MFRP基因的表达。在制备转基因小鼠的实验中，采用了两个转基因基因的Col2a1-FLAG-MFRP，以便于利用FLAG表位标签检测基因产物。将Col2a1-FLAG-MFRP注射到DBA/1(CRJ)小鼠胚胎中，并将形态完整的胚胎移植到ICR小鼠的输卵管中。将344枚转基因胚胎中的310枚移植给11只小鼠，其中2只受孕并产下4只F0代小鼠。用FLAG-MFRP特异性引物对基因组DNA进行RT-PCR分析，仅在一只F0小鼠中检测到转基因。这只转基因阳性的F0小鼠与野生型小鼠的表型几乎相同。我们现在正在通过将这只F0小鼠与野生DBA/1(CRJ)小鼠杂交来培育F1小鼠，并分析出生的53只F1小鼠中的转基因。作为对照研究，我们用抗胶原蛋白的单抗(致炎单抗鸡尾酒，IBL)在野生型小鼠上进行了关节炎诱导实验，并成功地在所有受试小鼠中引起了关节炎。今后，我们将继续对胚胎进行转基因注射，以获得更多的转基因小鼠候选品系。这个项目现在正在进行中。

项目成果

Yahata K., Mori K., Arai H., Koide S., Ogawa Y., Mukoyama M., Sugawara A., Ozaki S., Tanaka I.and Nakao K.: "Molecular cloning and expression of a novel klotho-related protein."J.Mol. Med.. 78. 389-394 (2000)

Yahata K.、Mori K.、Arai H.、Koide S.、Okawa Y.、Mukoyama M.、Sukawara A.、Ozaki S.、Tanaka I. 和 Nakao K.：“新型 klotho- 的分子克隆和表达

尾崎承一(共著): "内科学"文光堂(東京)、黒川清・松澤佑次・主幹編集. 2233 (1999)

Shoichi Ozaki（合着）：《内科医学》文科堂（东京）、黑川清、松泽佑二主编2233（1999）。

Ochiai K.^*, Ozaki S.^*, Tanino A., Watanabe S., Ueno T., Mitsui K., Toei J., Inada Y., Hirose S., Shirai T.and Nishimura H.(^* the first two authors contributed equally to this work): "Genetic regulation of anti-erythrocyte autoantibodies and splenomegal

落合 K.^*、尾崎 S.^*、谷野 A.、渡边 S.、上野 T.、三井 K.、东映 J.、稻田 Y.、广濑 S.、白井 T. 和西村 H.(^*

Tanaka M., Kishimura M., Ozaki S., Osakada F., Hashimoto H., Okubo M., Murakami M.and Nakao K.: "Cloning of novel soluble gp130 and detection of its neutralizing autoantibodies in rheumatoid arthiritis."J.Clin. Invest.. 106 (1). 137-144 (2000)

Tanaka M.、Kishmura M.、Ozaki S.、Osakada F.、Hashimoto H.、Okubo M.、Murakami M.和 Nakao K.：“类风湿性关节炎中新型可溶性 gp130 的克隆及其中和自身抗体的检测。”J

Ma W., Ozaki S., Sobajima J., Uesugi H., Murakami M., Tanaka M., Kozuki M., Hashimoto H., Fujita Y., Kawabata D., Osakada F., Shirakawa H., Yoshida M., Hayami M.and Nakao K.: "Detection of anti-neutrophil cytoplasmic antibodies in MRL/Mp-lpr/lpr mice and

Ma W.、Ozaki S.、Sobajima J.、Uesugi H.、Murakami M.、Tanaka M.、Kozuki M.、Hashimoto H.、Fujita Y.、Kawabata D.、Osakada F.、Shirakawa H.、Yoshida M