鸡生殖干细胞中Piwi基因的干涉效率  

Interference Efficiency of Piwi Gene Expression in the Chicken Germ Stem Cells

作　　者：李志腾[1] 常国斌[1] 徐璐[1] 马腾[1] 陈静[1] 陈蓉[1] 王洪志[1] 刘璐[1] 徐琪[1] 陈国宏[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009

出　　处：《中国农业科学》2016年第3期563-572,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31372297;31301966);国家科技支撑计划项目(2015BAD03B03)

摘　　要：【目的】针对禽类干细胞研究中,存在转染效率与干涉效率低下的问题,通过比较不同干涉方式对鸡原始生殖细胞(primordial germ cells,PGCs)中Piwi基因的干涉效果,探讨提高禽类生殖干细胞干涉效率的有效方法。为进一步研究Piwi基因参与干细胞自我更新、RNA沉默以及转录后调控过程等生物学功能研究奠定基础。【方法】根据已建立的原代细胞分离技术,分别从健康鸡群的10日龄和4日龄鸡胚中分离成纤维细胞(chicken embryo fibroblast,CEF)与生殖脊,其中CEF作为PGCs细胞的饲养层,生殖脊经Trypsin-EDTA室温下消化获得PGCs细胞,并通过形态学、化学方法(PAS糖原染色、AKP活性检测)和免疫学方法(TERT抗体、SSEA-1抗体)对其进行鉴定。根据Genbank公布的鸡Piwi基因的m RNA序列(NM_001098852),利用在线软件分别靶向不同位点设计合成得到3个小片段的stealth si RNAs,并将通用无义序列BLOCK-i T^(TM) Alexa Fluor®Red Fluorescent Oligo作为荧光素标记的阴性对照si RNA。同时,在线设计三条干扰序列和一条非特异性阴性对照序列,将其插入线性化空载体p RNA-U6.1,构建不同的sh RNA干涉载体。分别采用不同类型的转染试剂将构建好的si RNA和sh RNA转染PGCs细胞。首先利用通用无义si RNA以及仅带有GFP荧光标记基因的sh RNA载体作为阴性对照,检测si RNA和sh RNA转染效率,确定最佳转染条件。其次,将试验组si RNA和sh RNA在优化条件下转染PGCs细胞,分别收集转染了24、48、72和144 h这4个时间点的细胞,提取各个时间段的总RNA,采用实时荧光定量PCR技术检测Piwi基因m RNA表达水平,并用SPSS软件分析不同处理组的差异。【结果】在对原代分离的PGCs细胞进行了形态学、化学以及免疫学方法鉴定后,确认其所获得的细胞为PGCs细胞且状态良好。根据不同的转染试剂量与si RNA或sh RNA载体量的配比,得出si RNA转染最优条件为si RNA﹕Lipofectamine TM 2[Objective]Solving the low transfection efficiency and interference efficiency in poultry stem cell is a big challenge so far. This study was designed to compare the interference effects on Piwi gene expression in chicken primordial germ cells （PGCs） using different ways, aiming to explore effective methods to interfere the poultry stem cells, which is a fundamental work for the study of the self-renewal of stem cell, RNA silencing and post-transcriptional regulation. [Method] According to the techniques of the separation of primary germ cell, the authors selected 10th and 4th days fertilized eggs post incubation to separate the chicken embryo fibroblast cells（CEF） and germinal ridge, respectively; Herein, the CEF cells were taken as a feeder layer and the PGCs cells were treated by Trypsin-EDTA. The PGCs cells were further identified by the morphology, chemical method and immunology assay. According to the mRNA sequence of chicken Piwi gene published by Genbank （NM_001098852）, three positive siRNAs were chemically synthesized and the BLOCK-iTTM Alexa Fluor Red Fluorescent Oligo was as a negative control. Simultaneously, three interfering vectors of shRNA were constructed using the free carrier of pRNA-U6.1, and an empty vector with GFP fluorophore was as a negative control. Afterwards, the transfection efficiency was detected by the siRNA and shRNA negative control using different types of transfection reagent to optimize the condition, and extracted the RNA of PGCs cells at 24, 48, 72 and 144 h post transfection and analyzed differential expressions of Piwi gene with RT-PCR techniques. All data were analyzed by the SPSS software. [Result] With the premise of well identification of the PGCs cells, siRNA and shRNA were successfully transfected into the cells under the optimized conditions with 50 μmol siRNA and 2 μL Lipofectamine TM 2000 for siRNA transfection group and 500 ng shRNA and 1.5 μL X-Treme GENE HP DNA Transfection Reagent for shRNA transfection group. Compared to the blank group, the Pi

关 键 词：Piwi基因 siRNA SHRNA PGCS RNAI 

分 类 号：S831[农业科学—畜牧学]