Generation of genetically modified cell lines and mice using CRISPR/Cas9 system
Japan (IFMSA-Japan) - Hyogo Medical University, Hyogo(Kansai area)
Department of Genetics
Masaki Ohmuraya
Masaki Ohmuraya
4 weeks
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
No No No No No Yes No No No No No No
Type of Research Project
- Basic science
What is the background of the project?
The CRISPR/Cas9 system has been used for efficiently edit the genomes of diverse model organisms. Using CRISPR/Cas9 system, we have generated several gene modified cell lines and mice. CRISPR/Cas9 system is uncomplicated, economic, high throughput, and highly efficient in vitro and vivo genome editing and possibly in other mammals. We use this new technology to edit genomes of various cells and organisms, especially mice. We try to extract the DNA, RNA and protein from these cells and mouse organs and elucidate the role of each gene.
What is the aim of the project?
We are currently approaching a simple and economic electroporation–based strategy to deliver Cas9/sgRNA into cells and mouse zygotes. We have created various genetically modified cells and mice to elucidate the role of genes. In addition, we also investigate the relationship with illness.
What techniques and methods are used?
Molecular biology techniques. 1. Micropipetting 2. Plasmid DNA production 3. Polymerase Chain Reaction (PCR) 4. Purification of PCR products 5. Sequencing reaction 6. Western blot
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
What are the tasks expected to be accomplished by the student?
Master basic techniques such as Western blotting, PCR, and plasmid preparation. In the Western blot method 1. Sample adjustment 2. Electrophoresis 3. Transfer 4. Blotting 5. Shooting In the PCR method 1. Extraction of DNA from cells 2. Concentration measurement 3. Adjustment of reagents 4. Operation of PCR device 5. Electrophoresis Plasmid preparation 1. Cleavage of DNA 2. Ligation of different DNAs 3. Transformation into E. coli 4. Culture 5. Purification of plasmid DNA
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
The knowledge on molecular biology is required
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts:
- Medical students
- Inhibition of 15-PGDH causes Kras-driven tumor expansion through prostaglandin E2-ALDH1 signaling in the pancreas. Arima K; Ohmuraya M; Miyake K; Koiwa M; Uchihara T; Izumi D; Gao F; Yonemura A; Bu L; Okabe H; Imai K; Hashimoto D; Baba Y; Chikamoto A; Yamashita YI; Furukawa T; Araki K; Baba H; Ishimoto T. Oncogene. 2019 Feb;38(8):1211-1224. doi: 10.1038/s41388-018-0510-y. Epub 2018 Sep 24. PMID: 30250298
- Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome. Sakata K; Araki K; Nakano H; Nishina T; Komazawa-Sakon S; Murai S; Lee GE; Hashimoto D; Suzuki C; Uchiyama Y; Notohara K; Gukovskaya AS; Gukovsky I; Yamamura KI; Baba H; Ohmuraya M. Sci Rep. 2016 Nov 15;6:37200. doi: 10.1038/srep37200. PMID: 27845447