摘要
目的构建人nestin基因表达载体,制备nestin过表达转基因小鼠。方法以人神经胶质瘤细胞株U251的cDNA为模板,分3段扩增出nestin基因cDNA全序列,并将其克隆入含有强启动子ROSA26的pBROAD3载体,酶切鉴定、测序,除去原核序列,纯化。显微原核注射建立转基因小鼠。PCR验证nestin基因整合,确定建立首建者。将阳性鼠传代。Western blot方法检测F3代转基因小鼠与野生型小鼠主要器官(心、肺、脑、肾)的nestin表达。结果测序结果证明成功构建了受ROSA26启动子调控的nestin转基因载体。出生34只小鼠,PCR检测证实存在首建者2只。Western blot方法证实,与野生型小鼠相比,F3代转基因小鼠脑、肺组织存在较高水平nestin表达。结论首次成功建立nestin过表达转基因小鼠,外源基因可遗传到子代并在脑和肺组织中稳定表达,为深入研究nestin在肿瘤转移、细胞"干性"的维持和细胞的分化等功能提供了良好的实验动物模型。
Objective To develop a nestin transgenic mice and study the distribution of nestin expression in the organs.Methods The three segments of nestin in full-length cDNA was amplified using human glioma cell line U251 cDNA as the template and cloned into the vector pBROAD3 containing a strong promoter ROSA26.The constructed vector,after identification with restriction enzyme and sequencing and removal of the prokaryotic sequences,were purified and injected into the fertilized eggs of mice.Transgenic mice were identified by PCR and the founder was maintained.Western blot analysis was used to detect the expression of nestin of the F3 transgenic mice and the wild-type ones in the vital organs(heart,lung,brain and kidney).Results The Nestin transgenic vector controlled by ROSA26 promoter was successfully constructed and validated by sequencing.Among the 34 newborn mice,2 founders were tested to be nestin-positive by PCR.Westem blot analysis showed that the F3 transgenic mice expressed high levels of nestin in the brain and lungs.Conclusion Nestin transgenic mice have been successfully established with stable nestin expression in the brain and lungs of the offspring mice,which can be useful for studying the functions of nestin in tumor metastasis,stemness maintenance and differentiation of cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第8期1155-1159,共5页
Journal of Southern Medical University
基金
国家自然科学基金委与广东省政府联合重点基金(U0732006)~~
