鸡原始生殖细胞转染条件优化

doi:10.16288/j.yczz.20-212

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

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)

Abstract

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

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.

Figures/Tables 7

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Table 1

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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

Optimization of transfection conditions of chicken primordial germ cells

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