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

Research Project

Project/Area Number	10671644
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Ophthalmology
Research Institution	OKAYAMA UNIVERSITY
Principal Investigator	OHTSUKI Hiroshi Okayama Univ. Medical School, Professor, 医学部, 教授 (70093672)
Co-Investigator(Kenkyū-buntansha)	HASEBE Satoshi Okayama Univ. Medical School Hospital, Lecturer, 医学部・附属病院, 講師 (20263577) MATSUO Toshihiko Okayama Univ. Medical School Hospital, Lecturer, 医学部・附属病院, 講師 (90211565)
Project Period (FY)	1998 – 1999
Project Status	Completed (Fiscal Year 1999)
Budget Amount *help	¥3,000,000 (Direct Cost: ¥3,000,000) Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000) Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
Keywords	Strabismus / Extraocular muscle / cDNA / CDNA / 乳児内斜視
Research Abstract	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.