鸡原始生殖细胞转染条件优化

doi:10.16288/j.yczz.20-212

遗传 ›› 2021, Vol. 43 ›› Issue (3): 280-288.doi: 10.16288/j.yczz.20-212

• 技术与方法 • 上一篇

鸡原始生殖细胞转染条件优化

邹娴¹(), 何燕华¹, 何静怡¹, 王艳¹, 舒鼎铭¹(), 罗成龙¹()

¹ 广东省农业科学院动物科学研究所,畜禽育种国家重点实验室,广东省畜禽育种与营养研究重点实验室,广州 510640

收稿日期:2020-07-07 出版日期:2021-03-16 发布日期:2021-01-29
基金资助:
广东省自然科学基金项目(2018B030311044);,广东省科技计划项目(2017B020232003);,广东省科技计划项目(2019A050505007);,2020年省级现代农业科技联盟建设共性关键技术创新团队项目(2020KJ106);国家肉鸡产业技术体系岗位科学家项目(CARS-41);科技创新战略专项资金—高水平农科院建设项目(R2017PY-QY007)

Optimization of transfection conditions of chicken primordial germ cells

Xian Zou¹(), Yanhua He¹, Jingyi He¹, Yan Wang¹, Dingming Shu¹(), Chenglong Luo¹()

¹ State Key Laboratory of Livestock and Poultry Breeding & Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China

Received:2020-07-07 Online:2021-03-16 Published:2021-01-29
Supported by:
the National Natural Science Foundation of Guangdong Province(2018B030311044);the Science and Technology Program of Guangdong Province(2017B020232003);the Science and Technology Program of Guangdong Province(2019A050505007);2020 Provincial Modern Agricultural Science and Technology Alliance Construction of Common Key Technology Innovation Team Project(2020KJ106);the Earmarked Fund for Modern Agro-Industry Technology Research System(CARS-41);Special Fund for Scientific Innovation Strategy-construction of High Level Academy of Agriculture Science(R2017PY-QY007)

摘要/Abstract

摘要：

为获得鸡原始生殖细胞(primordial germ cells, PGCs)的最佳转染效率,本研究比较不同质粒用量和不同细胞数在3种转染试剂(Lipofectamine 2000、3000和LTX & Plus Reagent)中PGCs的转染效率,利用荧光激活细胞分选技术(fluorescence activated cell sorting technology, FACS)辅助优化Lipofectamine 3000转染试剂,经FACS进一步分选获得带绿色荧光蛋白(GFP)的PGCs,继续培养3周后,移植回注到受体鸡胚中,移植3.5 d后分离性腺拍照观察。结果显示,转染试剂Lipofectamine 3000的转染效率最高,质粒、Lipofectamine 3000转染试剂和PGCs细胞数的配比为3 μg: 4 μL: 0.5×10 ⁴个,转染5 h转染效率最高,达到23.4%,与现有的研究结果相比提高了2倍以上。移植回注PGCs到受体鸡胚中,荧光显微镜观察到鸡胚性腺中有GFP阳性细胞。本研究综合考虑转染试剂、质粒用量和细胞数量的影响因素以优化PGCs的转染条件,为高效制备转基因鸡及基因编辑鸡的研究奠定基础。

关键词: 鸡, 原始生殖细胞, 稳定转染, 脂质体

Abstract:

To improve the transfection efficiency of chicken primordial germ cells (PGCs), the present study evaluated the plasmid dosage and cell number on the efficiencies of three transfection reagents (Lipofectamine 2000, 3000 and LTX & Plus Reagent). PGCs was isolated from embryonic gonads of Huiyang bearded chicken. After 60 days of culture in vitro, the cells were transfected by using Lipofectamine transfection reagents with piggyBac vectors coding for the green fluorescence protein (GFP). PGCs were passaged in culture and fluorescent cells were screened and selected by flow cytometry at three days after transfection. At three weeks post transfection, about 2000 cells were injected into the stage 16 Hamburger and Hamilton (HH) embryos and incubated until stage 30 HH. The results showed that Lipofectamine 3000 was the best for transfection of PGCs. The highest transfection efficiency of PGCs could be achieved with a combination of 3 μg plasmid, 4 μL Lipofectamine 3000 transfection reagent and 0.5×10 ⁴PGCs cells. Flow cytometry analysis showed a 23.4% efficiency of stable transfection of PGCs using Lipofectamine 3000 with piggyBac vector, which was improved 2 times or more over current commonly used methods. After reinjecting PGCs into recipient chicken embryos, GFP-positive cells were observed in the gonads of the recipient chicken embryo by fluorescence microscopy. The study comprehensively evaluated the factors of transfection reagents, plasmid dosage and cell number to optimize the transfection of PGCs, thereby providing a foundation for the efficient preparation of transgenic and gene-edited chickens.

Key words: chicken, primordial germ cells, stable transfection, Lipofectamine

邹娴, 何燕华, 何静怡, 王艳, 舒鼎铭, 罗成龙. 鸡原始生殖细胞转染条件优化[J]. 遗传, 2021, 43(3): 280-288.

Xian Zou, Yanhua He, Jingyi He, Yan Wang, Dingming Shu, Chenglong Luo. Optimization of transfection conditions of chicken primordial germ cells[J]. Hereditas(Beijing), 2021, 43(3): 280-288.

图/表 7

图1

表1

图2

表2

图3

图4

图5

参考文献 21

[1]	Chojnacka-PuchtaL, SawickaD. CRISPR/Cas9 gene editing in a chicken model: current approaches and applications. J Appl Genet, 2020, 61(2): 221- 229.
[2]	ZhangG, LiC, LiQ, LiB, LarkinDM, LeeC, StorzJF, AntunesA, GreenwoldMJ, MeredithRW, ÖdeenA, CuiJ, ZhouQ, XuL, PanH, WangZ, JinL, ZhangP, HuH, YangW, HuJ, XiaoJ, YangZ, LiuY, XieQ, YuH, LianJ, WenP, ZhangF, LiH, ZengY, XiongZ, LiuS, ZhouL, HuangZ, AnN, WangJ, ZhengQ, XiongY, WangG, WangB, WangJ, FanY, DaFR, Alfaro-NúñezA, SchubertM, OrlandoL, MourierT, HowardJT, GanapathyG, PfenningA, WhitneyO, RivasMV, HaraE, SmithJ, FarreM, NarayanJ, SlavovG, RomanovMN, BorgesR, MachadoJP, KhanI, SpringerMS, GatesyJ, HoffmannFG, OpazoJC, HåstadO, SawyerRH, KimH, KimKW, KimHJ, ChoS, LiN, HuangY, BrufordMW, ZhanX, DixonA, BertelsenMF, DerryberryE, WarrenW, WilsonRK, LiS, RayDA, GreenRE, O'BrienSJ, GriffinD, JohnsonWE, HausslerD, RyderOA, WillerslevE, GravesGR, AlströmP, FjeldsåJ, MindellDP, EdwardsSV, BraunEL, RahbekC, BurtDW, HoudeP, ZhangY, YangH, WangJ, ConsortiumAG, JarvisED, GilbertMT, WangJ. Comparative genomics reveals insights into avian genome evolution and adaptation. Science, 2014, 346(6215):1311- 1320.
[3]	van de Lavoir MC, DiamondJH, LeightonPA, Mather-LoveC, HeyerBS, BradshawR, KerchnerA, HooiLT, GessaroTM, SwanbergSE, DelanyME, EtchesRJ. Germline transmission of genetically modified primordial germ cells. Nature, 2006, 441(7094):766- 769.
[4]	KoslováA, TrefilP, MucksováJ, ReinišováM, PlachýJ, KalinaJ, KučerováD, GerykJ, KrchlíkováV, LejčkováB, HejnarJ. Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus. Proc Natl Acad Sci USA, 2020, 117(4):2108- 2112.
[5]	LeeHJ, YoonJW, JungKM, KimYM, ParkJS, LeeKY, ParkKJ, HwangYS, ParkYH, RengarajD, HanJY. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development. FASEB J, 2019, 33(7): 8519- 8529.
[6]	DimitrovL, PedersenD, ChingKH, YiH, CollariniEJ, IzquierdoS, vande Lavoir MC, LeightonPA. Germline gene editing in chickens by efficient CRISPR-mediated homologous recombination in primordial germ cells. PLoS One, 2016, 11(4): e154303.
[7]	OishiI, YoshiiK, MiyaharaD, KagamiH, TagamiT. Targeted mutagenesis in chicken using CRISPR/Cas9 system. Sci Rep, 2016, 6: 23980.
[8]	ChenMJ, ChenDY, XieL, LuZP, YangMM, MoLF, LUKH, LuYQ. Effect of recipient embryos age on homing of chicken primordial germ cell after transplantation. Journal of Southern Agriculture, 2016, 47(06): 1014- 1018.
	陈美娟, 陈东阳, 谢龙, 陆振萍, 杨蒙蒙, 莫丽芬, 卢克焕, 陆阳清. 受体胚龄对鸡原始生殖细胞移植后归巢的影响. 南方农业学报, 2016, 47(06): 1014- 1018.
[9]	LillicoSG, McGrewMJ, ShermanA, SangHM. Transgenic chickens as bioreactors for protein-based drugs. Drug Discov Today, 2005, 10(3):191- 196.
[10]	IntarapatS, SternCD. Chick stem cells: Current progress and future prospects. Stem Cell Res, 2013, 11(3):1378- 1392.
[11]	LeeHJ, LeeHC, HanJY. Germline modification and engineering in avian species. Mol Cells, 2015, 38(9):743- 749.
[12]	MacdonaldJ, TaylorL, ShermanA, KawakamiK, TakahashiY, SangHM, McGrewMJ. Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci USA, 2012,109(23): E1466-E1472.
[13]	HeCW, HeYL, WuYH, ShaoFH. Effects of different transfection reagents on lentiviral packaging efficiency and the lentiviral infection efficiency of guinea-pig fibroblasts after packaging. Heilongjiang Anim Sci Veter Med, 2015, ( 10): 34- 38.
	何承文, 何玉龙, 吴月红, 邵风慧. 不同转染试剂对慢病毒包装及包装后病毒感染豚鼠成纤维细胞效率的影响. 黑龙江畜牧兽医, 2015, ( 10): 34- 38.
[14]	ZhongCL, LiGL, MoJX, QuanR, WangHQ, LiZC, WuZF, ZhangXW. Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators. Hereditas (Beijing), 2017, 39(10): 930- 938.
	钟翠丽, 李国玲, 莫健新, 全绒, 王豪强, 李紫聪, 吴珍芳, 张献伟. 不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响. 遗传, 2017, 39(10): 930- 938.
[15]	MacdonaldJ, GloverJD, TaylorL, SangHM, McGrewMJ. Characterisation and germline transmission of cultured avian primordial germ cells. PLoS One, 2010, 5(11): e15518.
[16]	ChoiJW, KimS, KimTM, KimYM, SeoHW, ParkTS, JeongJW, SongG, HanJY. Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells. PLoS One, 2010, 5(9): e12968.
[17]	ParkTS, HanJY. piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci USA, 2012, 109(24):9337- 9341.
[18]	YamamotoY, UsuiF, NakamuraY, ItoY, TagamiT, NirasawaK, MatsubaraY, OnoT, KagamiH. A novel method to isolate primordial germ cells and its use for the generation of germline chimeras in chicken. Biol Reprod, 2007, 77(1): 115- 119.
[19]	WangL, ChenMJ, ChenDY, PengSF, ZhouXL, LiaoYY, YangXG, XuHY, LuSS, ZhangM, LuKH, LuYQ. Derivation and characterization of primordial germ cells from Guangxi yellow-feather chickens. Poult Sci, 2017, 96(5):1419- 1425.
[20]	TaylorL, CarlsonDF, NandiS, ShermanA, FahrenkrugSC, McGrewMJ. Efficient TALEN-mediated gene targeting of chicken primordial germ cells. Development, 2017, 144(5):928- 934.
[21]	NaitoM, HarumiT, KuwanaT. Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens. Anim Reprod Sci, 2015, 153:50- 61.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

基因	引物序列(5′→3′)	扩增产物长度(bp)
POU5F1	CCACCCTCCCCACCTCTAC	181
POU5F1	TGTGCCAGCCTAACCTCCT	181
PRDM14	TGTTCGCCTACCGCTACTACCG	106
PRDM14	AGTGCTGGCGGAGTGTGTGTG	106
NANOG	AGACCTCTCCTTGACCACA	171
NANOG	CCACTCTCACCTTTATCCT	171
DDX4	TCCCAGAGCCCACACAGATG	124
DDX4	AAGTGATGCGCCCTCCTCTC	124
GAPDH	GGTGGTGCTAAGCGTGTTAT	151
GAPDH	ACCTCTGCCATCTCTCCACA	151

Lipofectamine 2000	Lipofectamine 3000	Lipofectamine LTX & Plus Reagent
A1 (20.30±1.70)^b	B1 (50.40±7.10)^c	C1 (30.00±5.50)^c
A2 (40.50±3.50)^a	B2 (102.10±3.10)^b	C2 (55.90±4.40)^b
A3 (46.50±5.10)^a	B3 (278.50±10.90)^a	C3 (80.90±8.10)^a
A4 (40.70±10.80)^a	B4 (75.20±8.60)^b	C4 (63.30±9.20)^b

鸡原始生殖细胞转染条件优化

Optimization of transfection conditions of chicken primordial germ cells

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 21

相关文章 15

编辑推荐

Metrics

[1]	高智慧, 黄佳新, 罗昊玉, 徐海冬, 娄明, 宁博林, 邢晓旭, 牟芳, 李辉, 王宁. 鸡NRG4基因组及转录本结构分析[J]. 遗传, 2023, 45(5): 447-458.
[2]	王冰源, 牟玉莲, 李奎, 刘志国. 农业动物干细胞研究进展[J]. 遗传, 2020, 42(11): 1073-1080.
[3]	冷奇颖, 郑嘉辉, 徐海冬, PatriciaAdu-Asiamah, 张颖, 杜炳旺, 张丽. 鸡胰岛素降解酶基因环状转录本克隆及其表达规律[J]. 遗传, 2019, 41(12): 1129-1137.
[4]	陈家辉, 任学义, 李丽敏, 卢诗意, 程湉, 谭量天, 梁少东, 何丹林, 罗庆斌, 聂庆华, 张细权, 罗文. 转录组测序揭示细胞周期通路参与鸡腹脂沉积[J]. 遗传, 2019, 41(10): 962-973.
[5]	褚衍凯,靳艳飞,邢天宇,马广伟,崔婷婷,闫晓红,李辉,王宁. 鸡PPARγ基因转录本3上游开放阅读框转录后的调控作用[J]. 遗传, 2018, 40(8): 657-667.
[6]	李敏,董翔宇,梁浩,冷丽,张慧,王守志,李辉,杜志强. 肉鸡腹脂率双向选择系群体表型数据库(NEAUHLFPD)的设计及其功能实现[J]. 遗传, 2017, 39(5): 430-437.
[7]	张潇飞,宋鹤,刘静,张文建,闫晓红,李辉,王宁. 鸡miR-17-92基因簇靶基因ZFPM2的鉴定及功能分析[J]. 遗传, 2017, 39(4): 333-345.
[8]	李光奇, 孙从佼, 吴桂琴, 石凤英, 刘爱巧, 孙皓, 杨宁. 利用转录组测序筛选鸡蛋褐壳性状相关基因[J]. 遗传, 2017, 39(11): 1102-1111.
[9]	程敏, 张文建, 邢天宇, 闫晓红, 李玉茂, 李辉, 王宁. 鸡miR-17-92基因簇上游调控区功能分析[J]. 遗传, 2016, 38(8): 724-735.
[10]	杨盛智, 吴国艳, 龙梅, 邓雯文, 王红宁, 邹立扣. 鸡蛋生产链中沙门氏菌对抗生素及消毒剂的耐药性研究[J]. 遗传, 2016, 38(10): 948-956.
[11]	张涛, 王文浩, 张跟喜, 王金玉, 薛倩, 顾玉萍. 京海黄鸡体重性状全基因组关联分析[J]. 遗传, 2015, 37(8): 811-820.
[12]	马俊平, 杨犀, 律娜, 刘飞, 陈燕, 朱宝利. 鸡T细胞受体γ链基因位点重新测序和组装[J]. 遗传, 2015, 37(6): 568-574.
[13]	宋晓燕, 张德祥, 张文武, 季从亮, 张细权, 罗庆斌. 鸡3种组织中热应激相关基因的表达谱芯片分析[J]. 遗传, 2014, 36(8): 800-808.
[14]	李金龙, 唐韶青, 邹智元, 王海潮, 徐青. 北京油鸡MSAP毛细管电泳荧光检测技术的建立[J]. 遗传, 2014, 36(5): 495-502.
[15]	牧仁，边艳超，浦亚彬，李向臣，王凤龙，关伟军. 北京油鸡胚胎肝脏来源间充质干细胞的分离培养及生物学特性[J]. 遗传, 2013, 35(3): 365-372.