摘要
为了制备小鼠 β-1,4-半乳糖基转移酶 ( β-1,4-galactosyltransferase ,β-1,4-Gal T- )地高辛标记的 RNA探针以探讨β-1,4-Gal T- m RNA在坐骨神经组织的表达定位 ,本研究用提取的小鼠脑总 RNA,采用 RT-PCR方法 ,得到β-1,4-Gal T- 的 DNA片断 ,将其克隆到 p GEM-T载体 ;采用体外转录的方法合成地高辛标记的正、反义β-1,4-Gal T- RNA探针 ;运用点杂交的方法分析标记探针的灵敏度 ;最后应用该探针 ,通过原位杂交的方法 ,分析 β-1,4-Gal T- m RNA在小鼠坐骨神经的表达。结果 :构建了β-1,4-Gal T- /p GEM-T质粒 ,获得了高效价的地高辛标记的正、反义β-1,4-Gal T- RNA探针 ,应用该探针发现 β-1,4-Gal T- 在小鼠坐骨神经髓鞘中有表达。以上结果表明 ,制备的地高辛标记的正、反义 β-1,4-Gal T- RNA原位杂交探针可特异地检测β-1,4-Gal T- m RNA在组织中的表达。本研究为进一步分析β-1,4-Gal T- 在坐骨神经及其它神经组织发育和损伤过程中的表达奠定基础。
In order to study the expression locations of β-1, 4-galactosyltransferase Ⅰ(β-1, 4-GalT-Ⅰ),the sense and anti-sense RNA probes for in situ hybridization of β-1, 4-galactosyltransferase Ⅰ were prepared in this study. The fragment of β-1, 4-GalT-Ⅰ was obtained by RT-PCR through total RNA of mouse brains. Amplified cDNA fragment was subcloned into pGEM-T Easy Vector, and plasmids were linearized with the restriction enzymes Apa Ⅰ and Sac Ⅰ. The digoxigenin (DIG) labeled sense and antisense probes were produced by using SP6 and T7 RNA polymerase respectively and in vitro transcription according to its protocol. Certificated by titer measuring and specificity analysis, the sense and anti-sense RNA probes were prepared successfully. The sense and anti-sense RNA probes for in situ hybridization of β-1, 4-galactosyltransferase Ⅰ were prepared in this experiment, which will provide an approach to study further the location of β-1, 4-GalT-Ⅰ particularly in nerve tissues.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2003年第4期365-368,共4页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金 ( No.3 0 3 0 0 0 99)
江苏省自然科学基金 ( BK2 0 0 3 0 3 5 )
江苏省教育厅自然科学项目 ( O3 KJB180 10 9)资助项目
