中国农业科学Issue(3)：563-572,10.DOI:10.3864/j.issn.0578-1752.2016.03.014
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
摘要
Abstract
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-iT™ 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 pmol siRNA and 2μL Lipofectamine TM 2000 for siRNA transfection group and 500 ng shRNA and 1.5μL X-Treme GENE HP DNA Transfection Reagent for shRNA transfection group. Compared to the blank group, thePiwi expressions of the negative control had no significant difference at the four time points (24,48,72 and 144 h) in siRNA and shRNA intervention group (P>0.05); Compared to the negative control and the blank group, Piwi gene expressions were significantly down-regulated in experimental groups at the first three time points in siRNA intervention group, while were significantly down-regulated at all of time points in the shRNA intervention group (P<0.05).[Conclusion]Collectively, the interference effects on Piwi gene expression in chicken PGCs were compared by siRNA and shRNA RNAi technology, and indicated that the shRNA vector had higher and more stable interference efficiency, which help to provide some basic information for future research of RNAi technology.关键词
Piwi 基因/siRNA/shRNA/PGCs/RNAiKey words
Piwi gene/siRNA/shRNA/PGCs/RNAi引用本文复制引用
李志腾,陈国宏,常国斌,徐璐,马腾,陈静,陈蓉,王洪志,刘璐,徐琪..鸡生殖干细胞中Piwi基因的干涉效率[J].中国农业科学,2016,(3):563-572,10.基金项目
国家自然科学基金（31372297，31301966）、国家科技支撑计划项目 （）
