Park, Doori
(Institute of Green Bio Science and Technology, Seoul National University)
,
Kim, Dongin
(National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University)
,
Jang, Green
(National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University)
,
Lim, Jongsung
(National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University)
,
Shin, Yun-Ji
(Natural Bio-Materials Inc.)
,
Kim, Jina
(Natural Bio-Materials Inc.)
,
Seo, Mi-Seong
(Natural Bio-Materials Inc.)
,
Park, Su-Hyun
(Crop Biotech Institute, Green-Bio Science and Technology, Seoul National University)
,
Kim, Ju-Kon
(Crop Biotech Institute, Green-Bio Science and Technology, Seoul National University)
,
Kwon, Tae-Ho
(Natural Bio-Materials Inc.)
,
Choi, Ik-Young
(Institute of Green Bio Science and Technology, Seoul National University)
Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effecti...
Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (${\sim}72{\times}coverage$) was sequenced with a $2{\times}150bp$ paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.
Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (${\sim}72{\times}coverage$) was sequenced with a $2{\times}150bp$ paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
, San Diego, CA, USA). In this pilot study, we demonstrated the potential of NGS for examination of transgenic insertion loci and discuss some technical bottlenecks of this new method.
대상 데이터
Thirteen PCR primers designed based on mapping direction validated the mapping results of 10 transgenic insert candidates. PCR results confirmed the target EGF sequence was successfully inserted on rice chromosome 4 (Figs.
성능/효과
Results were successful in differentiating NGS read types using in silico analyses from GM rice, PJKS131-2 and hypothetically, the outcome was acceptable in terms of read classification. However, as a validation step, we experienced unexpected problems.
참고문헌 (14)
1 Sabalza M Christou P Capell T Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks Biotechnol Lett 2014 36 2367 2379 25048244
2 Schürch C Blum P Zülli F Potential of plant cells in culture for cosmetic application Phytochem Rev 2008 7 599 605
3 Cohen SN Chang AC Boyer HW Helling RB Construction of biologically functional bacterial plasmids in vitro Proc Natl Acad Sci U S A 1973 70 3240 3244 4594039
4 European Parliament Commission Implementing Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. European Union Legislation. OJ L 268, 1-23 2003
5 European Parliament REGULATION (EC) No 1830/2003 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC. European Union Legislation. OJ L 268, 24-28 2003
6 European Parliament Commission Implementing Regulation (EU) No 503/2013 of 3 April 2013 on applications for authorisation of genetically modified food and feed in accordance with Regulation (EC) No 1829/2003 of the European Parliament and of the Council and amending Commission Regulations (EC) No 641/2004 and (EC) No 1981/2006. European Union Legislation. OJ L 157, 1-48 2013
7 Food and Agricultural Organization of the United Nations (FAO) Rep. No. CAC/GL 45-2003 Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants Rome FAO 2003
8 Food and Agricultural Organization of the United Nations (FAO) Rep. No. CAC/GL. 68-2008 Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants Rome FAO 2003
9 Yang L Wang C Holst-Jensen A Morisset D Lin Y Zhang D Characterization of GM events by insert knowledge adapted re-sequencing approaches Sci Rep 2013 3 2839 24088728
10 Pauwels K De Keersmaecker SC De Schrijver A du Jardin P Roosens NH Herman P Next-generation sequencing as a tool for the molecular characterisation and risk assessment of genetically modified plants: added value or not Trends Food Sci Technol 2015 45 319 326
11 Kovalic D Garnaat C Guo L Yan Y Groat J Silvanovich A The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology Plant Genome 2012 5 149 163
12 Wahler D Schauser L Bendiek J Grohmann L Next-generation sequencing as a tool for detailed molecular characterisation of genomic insertions and flanking regions in genetically modified plants: a pilot study using a rice event unauthorised in the EU Food Anal Methods 2013 6 1718 1727
13 Chan MT Chang HH Ho SL Tong WF Yu SM Agrobacterium -mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene Plant Mol Biol 1993 22 491 506 8392395
14 Goodstein DM Shu S Howson R Neupane R Hayes RD Fazo J Phytozome: a comparative platform for green plant genomics Nucleic Acids Res 2012 40 D1178 D1186 22110026
※ AI-Helper는 부적절한 답변을 할 수 있습니다.