Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (3): 563-472.doi: 10.3864/j.issn.0578-1752.2016.03.014

• ANIMAL SCIENCE·VETERINARY SCIENCERE • Previous Articles     Next Articles

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

LI Zhi-teng, CHANG Guo-bin, XU Lu, MA Teng, CHEN Jing, CHEN Rong, WANG Hong-zhi, LIU Lu, XU Qi, CHEN Guo-hong   

  1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2015-06-05 Online:2016-02-01 Published:2016-02-01

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 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 Piwi 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.

Key words: chicken, poultry stem cell ; primordial germ cells(PGCs), Piwi gene, siRNA, shRNA, RNAi

[1]    Cox D N, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily conserved genes defined by Piwi are essential for stem cell self-renewal. Genes & development, 1998, 12(23):3715-3727.
[2]    Cox D N, Chao A, Lin H. Piwi encodes a nucleoplasm factor whose activity modulates the number and division rate of gremlins stem cells. Development, 2000, 127(3):503-514.
[3]    李碧春, 施青青, 孙敏. 鸡生殖干细胞研究现状与展望. 中国家禽, 2011, 33(11):1-6.
Li B C, Shi Q Q, Sun M. The current status and prospects in germline stem cell. China Poultry, 2011, 33(11):1-6.(in Chinese)
[4]    陈胜锋, 王丙云, 计慧琴.鸡胚胎干细胞及其应用研究进展.动物医学进展, 2006, 27(1):9-13.
Chen S F, Wang B Y, Ji H Q. Applications and advances in chicken embryonic stem cells. Progress in Veterinary Medicine, 2006, 27(1): 9-13.(in Chinese)
[5]    Peters L, Meister G. Argonaute proteins: mediators of RNA silencing. Molecular Cell, 2007, 26(5):611-623.
[6]    Seto A G, Robert E, Lau N C. The coming of age for Piwi proteins. Molecular Cell, 2007, 26(8):603-609.
[7]    谢小燕, 裴雪涛. Piwi家族:干细胞分裂中的重要调控基因.生理科学进展, 2003, 34(2):139-141.
Xie X Y, Pei X T. Piwi family: An important gene regulate the stem cell division. Progress of Physiological Sciences, 2003, 34(2): 139-141. (in Chinese).
[8]    Kuramochi-miyagawa S, Kimura T, Ljirit W, Isobe T, Asada N, Fujita Y, Ikawa M, Iwai N, Okabe M, Deng W, Lin H, Matsuda Y, Nakano T. Mili, a mammalian member of Piwi family gene, is essential for spermatogenesis. Development, 2004, 131:839-849.
[9]    Deng W, Lin H. miwi, a murine homolog of Piwi, encodes a cytoplasmic protein essential for spermatogenesis. Developmental Cell, 2002, 2(6):819-830.
[10]   Shen X Y, Liu Y X, Chen H, Zhu S, Zhong X. Correlation between PIWI proteins and prognosis of Colon cancer. Chinese Journal of Gastrointestinal Surgery,2013, 18(9):530-535.
[11]   Paddison P J, CaudyA, Hannon G J. Stable suppression of gene expression by RNAi in mammalian cells. Science, 2002, 99(3): 1443-1448.
[12] Elbashir S M, Martinez J, Pakaniowska A, Lendeckel W, Tuschl T. Functional anatomy of siRNA for mediating efficient RNAi in Drosophila Melanogaster embryolysate. Embo Journal, 2001, 20: 6877-6888.
[13]   Holen T, Amarzguioui M A, Wiiger M T,  Babaie E,  Prydz H . Postional effect of short interferencing RNAs targetingthe human coagulation trigger tissue factor. Nucleic Acid Research, 2002, 30(8): 1757-1766.
[14]   秦玉新, 蒙凌华, 丁健. RNA干扰技术的研究进展. 中国药理学通报, 2007, 23(4):421-424.
Qin Y X, Meng L H, Ding J. Research progresses of RNA interference. Chinese Pharmacological Bulletin, 2007, 23(4):421-424. (in Chinese)
[15]   Tolia N H, Joshua T L. Slicer and the argonautes. Nature Chemical Biology, 2007, 3(1):36-43.
[16]   Hamburger V, Hamilton H L. A series of normal stages in the development of the chick embryo. Developmental Dynamics,1992, 195(4):231-272.
[17]   Li B C, Tian Z Q, Sun M, Xu Q, Wang X Y, Qin Y R. Directional differentiation of chicken primordial germ cells into adipocytes, neuron-like cells, and osteoblasts. Molecular Reproduction and Development, 2010, 77(9):795-801.
[18]   李建超, 张颖, 戴爱琴, 王洪志, 翟飞, 华登科, 夏明秀, 常国斌, 陈国宏. 体外抑制Piwi基因对鸡原始生殖细胞相关基因表达的影响.畜牧兽医学报, 2014, 45(6):871-878.
Li J C, Zhang Y, Dai A Q, Wang H Z, Zhai F, Hua D K, Xia M X, Chang G B, Chen G H. In Vitro inhibition of Piwi gene expression and the influence on related genes in primordial germ cells. Acta Veterinarian et Zootechnical Sinica, 2014, 45(6):871-878.(in Chinese)
[19]   Aravin A A, Hannon G J, Brennecke J. The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science, 2007, 318 (5851): 761-764.
[20]   任小青. RNA 干扰技术及其应用. 天津农学院学报, 2009, 16(1): 45-48.
Ren X Q. RNA Interference and its application. Journal of Tianjin
Agricultural University, 2009, 16(1): 45-48. (in Chinese)
[21]   房宝英, 何冬梅, 张洹. siRNA及其导入体内外方法的研究进展. 国际病理科学与临床杂志, 2007, 27(1):44-47.
Fang B Y, He D M, Zhang Y. SiRNA and its delivery in vivo and vitro. International Journal of Pathology and Clinical Medicine, 2007, 27(1):44-47. (in Chinese)
[22]   Ren Z, Li S, Wang Q L, Xiang Y F, Cui Y X, Wang Y F, Qi R B, Lu D X, Zhang S M, Zhang P Z. Effect of siRNA on HSV-1 plaque formation and relative expression levels of RR mRNA. Virologica Sinica, 2011, 26(1):40-46.
[23]   Paddison P J, Candy A A, Bernstein E, Bernstein E, Hannon G J, Conklin D S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cell. Genes & Development, 2002, 16: 948.
[24]   王燕丽, 郝柱, 李艳玲, 彭静, 沈一飞.梅岭土鸡原始生殖细胞的生物学特性. 农业生物技术学报, 2012, 20(4):397-403.
Wang Y L, Hao Z, Li Y L, Peng J, Shen Y F. Biological characterization of primordial germ cells from Meiling chicken (Gallus domesticus). Journal of Agricultural Biotechnology, 2012, 20(4):397-403.(in Chinese)
[25]   王红刚, 林波, 汪文利. 不同转染条件影响质粒转染效率的探讨. 河南大学学报, 2012, 31(2):111-118.
Wang H G, Lin B, Wang W L. Study for the influence of different transfected condition on plasmid transfected efficiency. Journal of Henan University, 2012, 31(2):111-118.(in Chinese)
[1] SHU JingTing,SHAN YanJu,JI GaiGe,ZHANG Ming,TU YunJie,LIU YiFan,JU XiaoJun,SHENG ZhongWei,TANG YanFei,LI Hua,ZOU JianMin. Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation [J]. Scientia Agricultura Sinica, 2022, 55(3): 589-601.
[2] ZHANG YaNan,JIN YongYan,ZHUANG ZhiWei,WANG Shuang,XIA WeiGuang,RUAN Dong,CHEN Wei,ZHENG ChunTian. Comparison of Shell Mechanical Property, Ultrastructure and Component Between Chicken and Duck Eggs [J]. Scientia Agricultura Sinica, 2022, 55(24): 4957-4968.
[3] TU YunJie,JI GaiGe,ZHANG Ming,LIU YiFan,JU XiaoJun,SHAN YanJu,ZOU JianMin,LI Hua,CHEN ZhiWu,SHU JingTing. Screening of Wnt3a SNPs and Its Association Analysis with Skin Feather Follicle Density Traits in Chicken [J]. Scientia Agricultura Sinica, 2022, 55(23): 4769-4780.
[4] HUANG XunHe,WENG ZhuoXian,LI WeiNa,WANG Qing,HE DanLin,LUO Wei,ZHANG XiQuan,DU BingWang. Genetic Diversity of Indigenous Yellow-Feathered Chickens in Southern China Inferred from Mitochondrial DNA D-Loop Region [J]. Scientia Agricultura Sinica, 2022, 55(22): 4526-4538.
[5] WANG ZhePeng,ZHOU WenXin,HE JunXi,HU QiaoYan,ZHAO JiaYue. Association of Levels of Cholecystokinin A Receptor Expression and Sequence Variants with Feed Conversion Efficiency of Lueyang Black-Boned Chicken [J]. Scientia Agricultura Sinica, 2022, 55(22): 4539-4549.
[6] GUO Jun,WANG KeHua,HAN Wei,DOU TaoCun,WANG XingGuo,HU YuPing,MA Meng,QU Liang. Analysis of Indirect Genetic Effects on Body Weight of 42 Day-Old Rugao Yellow Chickens [J]. Scientia Agricultura Sinica, 2022, 55(19): 3854-3861.
[7] YaTing JIA,HuiHui HU,YaJun ZHAI,Bing ZHAO,Kun HE,YuShan PAN,GongZheng HU,Li YUAN. Molecular Mechanism of Regulation by H-NS on IncFⅡ Plasmid Transmission of Multi-drug Resistant Chicken Escherichia coli [J]. Scientia Agricultura Sinica, 2022, 55(18): 3675-3684.
[8] GUAN RuoBing,LI HaiChao,MIAO XueXia. Commercialization Status and Existing Problems of RNA Biopesticides [J]. Scientia Agricultura Sinica, 2022, 55(15): 2949-2960.
[9] YIN Fei,LI ZhenYu,SAMINA Shabbir,LIN QingSheng. Expression and Function Analysis of Cytochrome P450 Genes in Plutella xylostella with Different Chlorantraniliprole Resistance [J]. Scientia Agricultura Sinica, 2022, 55(13): 2562-2571.
[10] WU Wei,XU HuiLi,WANG ZhengLiang,YU XiaoPing. Cloning and Function Analysis of a Serine Protease Inhibitor Gene Nlserpin2 in Nilaparvata lugens [J]. Scientia Agricultura Sinica, 2022, 55(12): 2338-2346.
[11] CHEN ErHu,MENG HongJie,CHEN Yan,TANG PeiAn. Cuticle Protein Genes TcCP14.6 and TcLCPA3A are Involved in Phosphine Resistance of Tribolium castaneum [J]. Scientia Agricultura Sinica, 2022, 55(11): 2150-2160.
[12] ZHANG NingBo,HAN ZhaoQing,JIN TaiHua,ZHUANG GuiYu,LI JiongKui,ZHENG QuanSheng,LI YongZhu. Comparison Analysis on Eggshell Quality, Biochemical Index of Calcium Metabolism and Calcium Binding Protein CaBP-D28k mRNA Expression Between Langya Chicken and Its Synthetic Lines [J]. Scientia Agricultura Sinica, 2021, 54(9): 2017-2026.
[13] WANG GuangYu,LI Qing,TANG WenQian,WANG HuHu,XU XingLian,QIU WeiFen. Effects of nuoB on Physiological Properties of Pseudomonas fragi and Its Spoilage Potential in Chilled Chicken [J]. Scientia Agricultura Sinica, 2021, 54(8): 1761-1771.
[14] Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG. Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids [J]. Scientia Agricultura Sinica, 2021, 54(6): 1143-1153.
[15] YuYan YANG,YaoWen LI,Shuang XING,MinHong ZHANG,JingHai FENG. The Temperature-Humidity Index Estimated by the Changes of Surface Temperature of Broilers at Different Ages [J]. Scientia Agricultura Sinica, 2021, 54(6): 1270-1279.
Full text



No Suggested Reading articles found!