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. [L. Genetics and genomics-5] Better expression methods of transposon system YunHaeng lee¹ ¹Protein engineering lab, incheon national university, incheon 22012, korea Among them, the transposon system has been found to be a reliable tool that enables the integration of
transgenes. In this study, we used the SB transposon vector and SB100X transposase vector needed to insert into the genome. This system showed better protein expression than the conventional vector system. Our study showed that ITR (IR / DR) was modified at both ends of the SB transposon to show higher protein expression than the existing SB transposon system. This was possible because the IR / DR size was small enough and was designed to have a small impact on the overall vector size. In addition, inhibition of DNA methylation by chemical treatment significantly improved protein expression by
preventing gene silencing that could occur in the SB transposon-mediated system. (Also, significant differences were found in the amount of transposon vector integration depending on the timing of methylation inhibitor treatment.) In conclusion, the results of the present invention are the first reports to show that the use of transposon systems has improved protein productivity in the past. In addition, the use of SB transposon systems developed in the biopharmaceutical industry will increase production efficiency. Cloning and functional characterization of insect odorant receptors 1, Je Jun
Oh 1,2 and Jun-Ho Lee * 1 Department of Biotechnology, Chonnam National University, Gwangju, Korea 2 Microzyme corporation, Damyang Samanli 234, Jeonmam, Korea Summary of the Drosophila Chromosome Screen ABSTRACT The highly specialized olfactory receptor neurons (ORNs) on the antennae of male moths can recognize blends of several pheromone components. In present study, a total of six candidate pheromone compounds are found and functionally characterized in the electophyological study. In here, we report on novel candidate pheromone compounds in the same species. The olfactory receptor is
analysed revealed that finding compounds are specifically affect in on olfactory receptor. In silico study revealed that odorant-gated ion channels comprised of a highly conserved co-receptor and our chemicals are binding at extra cellular site. Functional analyses on the odorant-gated ion channels comprised of a highly conserved co-receptor were then performed using the heterologous expression system of Xenopus oocytes. pheromone components did not respond to any tested pheromone components and analogs. These results may contribute to clarifying how pheromone detection works in odorant-gated
ion channels comprised of a highly conserved co-receptor. Key words: pheromone receptor; pheromone; Xenopus oocytes MATERIALS AND METHODS Cloning of Drosophila odorant-gated ion channels Recording and Data analysis Two-microelectrode voltage-clamp recording (Oocyte Clamp Adopted from Cell. 2017 Aug 10;170(4):736-747.e9. (OC-725C), Digidata 1200A) RESULTS Figure 1. Gene cloing of Drosophila odorant-gated ion channel (Orco) SUMMARY AND CONCLUSION In this study, whole cell voltage clamp recording was performed with cell expression system of OR65 gene, which is a subtype of olfactory
neuro-receptor isolated from Drosophila. After the successful expression of this receptor, microbial culture extract of microorganism, a harmful insect inducer, was used to investigate whether olfactory receptor DNA library activity was regulated. The activity of the receptor was confirmed in the microbial culture extract. Library of Drosophila odorant-gated ion channels The Scripps Research Institute (La Jolla, CA) Odorant receptor of Apocrypta bakeri, Protein Data Bank (ID A We show that it is possible to identify attractant or repellent code B0FAQ4, 3.5A resolution) substance using the
olfactory receptor activity regulating system of insects. MZ01 shows the attracting phenomenon by Figure 2. Drosophila odorant ion channel expression activating insect receptor OR65, The results of the scientific analysis of the performance of the extracts are presented. [L. Genetics and genomics-7] Cloning and functional characterization of insect odorant receptors Junho Lee¹ ¹Biotechnology, Chonnam National University, Gwangju 61186, Korea The highly specialized olfactory receptor neurons (ORNs) on the antennae of male moths can recognize blends of several pheromone components. In present
study, a total of six candidate pheromone compounds are found and functionally characterized in the electophyological study. In here, we report on novel candidate pheromone compounds in the same species. The olfactory receptor is analysed revealed that finding compounds are specifically affect in on olfactory receptor. In silico study revealed that odorant-gated ion channels comprised of a highly conserved co-receptor and our chemicals are binding at extra cellular site. Functional analyses on the odorant- gated ion channels comprised of a highly conserved co-receptor were then performed using
the heterologous expression system of Xenopus oocytes. pheromone components did not respond to any tested pheromone components and analogs. These results may contribute to clarifying how pheromone detection works in odorant- gated ion channels comprised of a highly conserved co-receptor. Molecular study of Olfactory Receptor 1, 1,2 Young Kun Shim , and Jun-Ho Lee * 1 Department of Biotechnology, Chonnam National University, Gwangju, Korea 2 Microzyme corporation, Damyang Samanli 234, Jeonmam, Korea ABSTRACT Concept of Experiments The olfactory nervous system recognizes and distinguishes many
different chemicals in the general living environment. Insects have evolved a group of odorant-gated ion channels composed of highly-developed olfactory receptors capable of distinguishing and distribution between various chemicals with symbolic or evasive specificities. Recently, aphid genomes related to olfaction, including olfactory receptors and proteins, have been identified and olfactory receptors have been reported that are differentially differentiated from Drosophila. The genome of the olfactory receptor has a very conservative sequence and a systematic signaling system. A
representative receptor, odorant-gated ion channels comprised of a highly conserved co-receptor (Orco) has a homotetramer channel structure with four subunits arranged symmetrically around the central hole. It has a very similar structure to the 7-transmembrane receptor present in the human body and has a very similar structural form and gating mechanism to receptors of neurotransmitters. In this study, whole cell voltage clamp recording was performed with cell expression system of OR65 gene, which is a subtype of olfactory receptor isolated from Drosophila. After the successful expression of
this receptor, microbial culture extract of microorganism, a harmful insect inducer, was used to investigate whether olfactory receptor activity was regulated. The activity of the receptor was confirmed in the recording media diluted 10,000 times with the microbial culture extract. Therefore, it is possible to identify attractant or repellent substance using the olfactory receptor activity regulating system of insects. Through this study, new attractant shows the attracting phenomenon by activating insect receptor OR65, The results of the scientific analysis of the performance of the extracts
are presented. Key words: olfactory nervous system
