Page 1 - L. Genetics and genomics
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Improved productivity of Sleeping Beauty transposon in CHO cell
Yun Haeng Lee and Joon Tae Park*
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, Korea
Tel: +82-32-835-8841
E-mail address: joontae.park@inu.ac.kr
A B
C
The eukaryotic genome contains a lot of repeat DNA, some of which show mobility [1].
Representative transposon mediator systems include Piggy Bac (PB), Tol2 and
Sleeping Beauty (SB) transposon [2]. These transposon systems use a cut and paste
method to bind the transposase protein to the region of the inverted terminal repeat 8
sequence (ITRs), cut the gene of interest and integrate it into genomic DNA [3]. D 6 *** *** E 10 *** ***
Relative copy number 4
Luciferase 8 6
β-actin 2 *** Relative mRNA expression 4 ***
0 2
2.0μg Ctrl V + - - - 0
So far, the SB transposon system has been used for many experiments and 2.0μg SB V - + - 2.0μg Ctrl V + - + - - -
2.0μg SB V
2.0μg - - + 2.0μg
productions, but has focused on reducing the vector size and developing the Developed SB V Developed SB V - - +
0.66μg SB100X - + + 0.66μg SB100X - + +
transposase of the SB transposon system [9]. Because conventional SB transposon transposase vector transposase vector
has low stability, the transgene that has been integrated over time is cut-out, and the Figure 3. Comparison of protein expression efficiency between conventional SB
protein expression decreases. We approached this problem. Our method focused on vector and developed SB vector system.
the modification of the SB transposon ITRs and created a new vector system. In
addition, gene silencing was inhibited by treatment with methylation inhibitor. Through
the two methods above, SB transposon system was created with better productivity. ***
This study has made large progress over the existing SB transposon system, which is A 2.010 7 B
anticipate to make a large difference in the biopharmaceutical market beyond simple 1.510 7
Luciferase 1.010 7 ***
research. ***
5.010 6
0
2.0μg Ctrl V + + - - - -
2.0μg SB V - - + + - -
2.0μg -
Developed SB V - - - + +
0.66μg SB100X - - + + + +
CMV SV40 transposase vector
Methylation
SP Luciferase NeoR Luciferase Ctrl inhibitor - + - + - +
SV40
Figure 4. Changes of gene copy number and luciferase productivity after
methylation inhibitor treatment.
CMV Conventional
IR/DR left arm SP Luciferase NeoR IR/DR right arm
SB vector(VTp)
The developed SB transposon vector modified with the same ITRs twice repeated
sequence showed a higher integration pattern than the conventional SB
transposon vector and expressed the protein more stably for a longer period.
CMV SV40 Developed When methylation inhibitor was treated in CHO cells to which SB transposon was
SP Luciferase NeoR
IR/DR dual left IR/DR dual right SB vector(VdTp) applied, gene silencing was inhibited and protein expression was increased.
arm arm
Based on the number of gene copies when methylation inhibitor was treated, it
was proved once again that the developed SB transposon vector was more stable.
Transposase
CMV
SB100X vector
Jin Z, Maiti S, Huls H, Singh H, Olivares S, Mátés L, Izsvák Z, Ivics Z, Lee DA,
Champlin RE, Cooper LJN: The hyperactive Sleeping Beauty transposase
SB100X improves the genetic modification of T cells to express a chimeric
antigen receptor. Gene therapy 2011, 18:849-856.
Balasubramanian S, Rajendra Y, Baldi L, Hacker DL, Wurm FM: Comparison of
three transposons for the generation of highly productive recombinant
CHO cell pools and cell lines. Biotechnol Bioeng 2016, 113:1234-1243.
1,000,000 *** 8.010 6
A *** B Balasubramanian S, Matasci M, Kadlecova Z, Baldi L, Hacker DL, Wurm FM:
800,000
6.010 6 Rapid recombinant protein production from piggyBac transposon-
Luciferase 600,000 Luciferase 4.010 6 mediated stable CHO cell pools. J Biotechnol 2015, 200:61-69.
400,000
***
2.010 6 Razin A, Cedar H: DNA methylation and gene expression. Microbiological
200,000 reviews 1991, 55:451-458.
0 1 2 3
2.0μg Ctrl V + - - 2.0 ug Ctrl V 0 + - -
2.0μg SB V - + + 2.0 ug SB V - + +
0.66μg SB100X - - + 0.66 ug SB100X
transposase vector transposase vector - - +
Luciferase
β-actin This research was supported by Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT
& Future Planning (NRF-2018R1D1A1B07040293) and a Chonnam National
Figure 2. Effect of SB transposon system for protein production. University R&D Program Grant for Research Chair Professors.
