Construction and Functional Verification of a Novel Two-in-one PiggyBac Transposon System

doi:10.13523/j.cb.2203005

China Biotechnology

2022, Vol. 42

Issue (6): 39-46 DOI: 10.13523/j.cb.2203005

Construction and Functional Verification of a Novel Two-in-one PiggyBac Transposon System

ZHANG Yao-cang¹,PENG Yu¹,DING Ke-xin²,TAI Fu-min²,ZHENG Xiao-fei²,QIN Yi-de¹(

),FU Han-jiang^1,²(

)

1. School of Basic Medicine, Anhui Medical University, Hefei 230032, China
2. Beijing Key Laboratory of Radiobiology, Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China

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Abstract

Objective: The PiggyBac (PB) transposon is a mobile genetic element that transposes between vectors and chromosomes using a “cut and paste” mechanism. Transposon elements and transposase expression cassettes were integrated into an expression vector to construct an easy-to-use two-in-one PB transposon system. Methods: The transposon elements and transposase expression cassette required for the PiggyBac transposon system were obtained by polymerase chain reaction (PCR), and the transposase expression cassette was recombined with the original pUC18 vector using T4 DNA ligase. Gibson homologous recombination technology was used to combine transposon elements with recombination vector to construct a two-in-one PB transposon system. The system was tested for its efficacy and reliability using enhanced green fluorescent protein (EGFP) and functional damage suppressor protein (DSUP). Results: EGFP was visible and bright in all puromycin-resistant cells. In addition, a cell line stably expressing functional DSUP was obtained through the two-in-one PB transposon system, demonstrating that the foreign gene can be efficiently integrated into the genomic DNA and expressed. Conclusion: A new two-in-one PB transposition system was successfully constructed, which made the establishment of stable expression cell lines more convenient and economical.

Key words： PiggyBac transposon Stable transfection EGFP

Received: 03 March 2022 Published: 07 July 2022

ZTFLH:

Q789

Corresponding Authors: Yi-de QIN,Han-jiang FU E-mail: fuhj75@126.com;yideqin@ahmu.edu.cn

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	Yao-cang ZHANG
	Yu PENG
	Ke-xin DING
	Fu-min TAI
	Xiao-fei ZHENG
	Yi-de QIN
	Han-jiang FU

Cite this article:

ZHANG Yao-cang,PENG Yu,DING Ke-xin,TAI Fu-min,ZHENG Xiao-fei,QIN Yi-de,FU Han-jiang. Construction and Functional Verification of a Novel Two-in-one PiggyBac Transposon System. China Biotechnology, 2022, 42(6): 39-46.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2203005 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I6/39

Table 1 Primers sequence for PCR

Fig.1 A flow diagram outlining the construction of the two-in-one PiggyBac transposon system

Fig.2 Colony polymerase chain reaction (PCR) validation of the vector(a) Validation of PB-All-EGFP-PuroR vector by colony PCR (b) Validation of PB-All-DUSP-PuroR by colony PCR vector (WT)

Fig.3 Usability verification of the two-in-one PiggyBac transposon system(a) The pEGFP-PuroR vector contains enhanced green fluorescent protein (EGFP) and puromycin resistance protein (PuroR) coding regions, the PB-All-EGFP-PuroR vector contains both EGFP-PuroR and transposase cassettes separated by 5' and 3' PiggyBac inverted terminal repeats (b) EGFP transient expression in HEK-293T cells transfected with the pEGFP-PuroR and PB-All-EGFP-PuroR vectors (c) EGFP stable expression in HEK-293T cells transfected with the pEGFP-PuroR and PB-All-EGFP-PuroR vectors. The stably expressing cells were selected by treating the cells with 2 μg/mL of puromycin for 14 days

Fig.4 Detection of inserts in genomic DNA

Fig.5 Stably expressing dual-specific phosphatase (DSUP) HEK-293T cell lines established using the all-in-one PiggyBac transposon system(a)Western blot indicated stable DSUP expression in HEK-293T cells (b)DSUP reduced the level of phosphor-histone-H2AX induced by γ-rays (c)DSUP decreased the γ-H2AX foci induced by γ-rays (d)Stably expressing DSUP counteracted the γ-ray-induced inhibition of HEK-293T cell proliferation. Data were presented as the mean ± s.e.m. of three independent experiments, ^*P<0.05 vs indicated group


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