Isolation of cDNA Clones Specific to the Estraocular Muscle of the Infantile Esotropia

婴儿内斜视眼肌特异性 cDNA 克隆的分离

基本信息

批准号：
10671644
负责人：
OHTSUKI Hiroshi
金额：
$ 1.92万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671644/
关键词：
Strabismus Extraocular muscle cDNA CDNA 乳児内斜視

项目摘要

To elucidate the underlying mechanism of infantile esotropia, we tried to determine mRNA which was expressed specifically in the extraocular muscle in patients with infantile esotropia. Fragments of the extraocular muscle excised during strabismus surgery in patients with infantile esotropia or intermittent exotropia were immediately frozen in liquid nitrogen and stored at -135C until use. The excised fragments were from the lateral rectus muscle in patients with infantile esotropia, while from the medial rectus muscle in patients with intermittent exotropia. The muscle fragments were pulverized in liquid nitrogen, and mRNA was isolated, followed by synthesis of cDNA. Subtractive hybridization based on suppression polymerase chain reaction (PCR) was used to isolate cDNAs which were expressed selectively in muscle fragments of infantile esotropia, compared with muscle fragments of intermittent exotropia. The cDNA were cloned into pCRII-TOPO vector. Dot-blot morthern blot analysis was used to examine the expression of isolated cDNA clones in muscle fragments of infantile esotropia or intermittent exotropia. We found that DNA smear, extending from 100 to 500 base pairs, was amplified by nested PCR from suppression PCR products after two rounds of hybridization. The smear DNA was cloned, and 240 plasmic clones were obtained. Dot-blot northern blot analysis confirmed that 10 clones were selectively expressed in muscle fragments of infantile esotropia, compared with muscle fragments of intermittent exotropia. Sequencing of these clones and GenBAnk search revealed that the sequence had no match to the known cDNA.

为了阐明婴儿内斜视的潜在机制，我们试图确定婴儿内斜视患者眼外肌中特异性表达的 mRNA。将婴儿内斜视或间歇性外斜视患者斜视手术中切除的眼外肌碎片立即冷冻在液氮中，并保存在-135°C直至使用。婴儿内斜视患者切除的碎片来自外直肌，间歇性外斜视患者切除的碎片来自内直肌。将肌肉碎片在液氮中粉碎，分离mRNA，然后合成cDNA。基于抑制聚合酶链反应（PCR）的消减杂交被用来分离在婴儿内斜视的肌肉片段中选择性表达的cDNA，与间歇性外斜视的肌肉片段相比。将cDNA克隆到pCRII-TOPO载体中。斑点印迹morthern印迹分析用于检查婴儿内斜视或间歇性外斜视肌肉片段中分离的cDNA克隆的表达。我们发现，经过两轮杂交后，通过巢式PCR，从抑制PCR产物中扩增出100至500个碱基对的DNA涂片。对涂片DNA进行克隆，获得240个质克隆。斑点印迹 Northern 印迹分析证实，与间歇性外斜视的肌肉片段相比，有 10 个克隆在婴儿内斜视的肌肉片段中选择性表达。对这些克隆进行测序和 GenBAnk 搜索表明该序列与已知的 cDNA 不匹配。