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Engineering Stem Cells via Molecular Gene Editing and Protein Biochemistry for clinical applications
Turkey (TurkMSIC) - Istinye University, Istanbul
Istinye University School of Medicine Histology & Embryology Dept. Edirne Cirpici Yolu No:9 , 34025. Cevizlibag / Istanbul / TURKEY
Prof. Dr. Erdal KARAOZ
Asst. Prof. Ayca Zeynep Ilter, Asst. Prof. Ozgur Tataroglu
Type of Research Project
- Basic science
What is the background of the project?
Istinye University Stem Cell and Tissue Engineering Research and Practice Center (ISUKOK) was established in 2017 and started its operations. In our stem cell laboratory, multipotent and pluripotent cells are cultured and can be monitored live with the latest technology microscopes. In addition, the center supports student projects and also provides internship and observation opportunities for students. It is a well-equipped laboratory where students interested in science can learn and apply simple techniques under the supervision of researchers. In this project students will learn to create their own cell lines using fibroblast and mesenchymal cells. In addition, students will also use molecular and biochemical tecniques such as western blotting, DNA and protein isolation and Real Time Polymerase Chain Reaction. In our university’s Histology & Embryology Department there are currently 7 Histology & Embryology specialists working. In Turkey there is also a society called “Turkish Histology and Embriology Society” that was established in 1990. This society does various actions based on Histology and Embriology like organizing National and International congress.
What is the aim of the project?
Istinye Stem Cell Lab is a histology and embriology institute with experimental studies on the treatment of Multiple Sclerosis, Parkinson’s Disease and demyelinating spinal cord injuries. The internship aims to acquaint students with the research procedures that involve use of mesenchymal stem cells with a focus on neuronal regenaration.
What techniques and methods are used?
Besides of cell culture techniques, there are also several molecular biology techniques such as agarose gel electrophoresis and Western Blot that are used frequently. Gel electrophoresis and Western Blot are used to analyze cell contents such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and proteins.Most widely used gel electrophoresis methods agarose gel electrophoresis and polyacrylamide gel electrophoresis. Generally, agarose gel is used for nucleic acids and polyacrylamide gel is used for proteins. SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE) immediately an be applied in the analysis of all protein types. We use agarose gel electrophoresis to make DNA classification. The stained DNA molecules are run on agarose gel to check the DNA quality. The method used for protein classification is western blotting. They work with the same mechanism. We use weights for proteins while we use lengths in DNA classification.And for western blotting tecnique; A plurality of different lysis buffers may be used, depending on the characteristics of the tissue in which the prototype of interest is present, or the cellular region or protein of the protein. After in vivo experiments with experimental animals, brain samples are generally homogenized with RIPA buffer (Radioimmunoprecipitation assay buffer). Weigh the tissue sample and add 10 times the volume of buffer. The protease inhibitor is added freshly to RIPA. Depending on whether the protein to be studied is phosphorylated, the content of the protease inhibitor should be changed and, where necessary, a phosphotase inhibitor should be used.
What is the role of the student?
- The student will mainly observe
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Learn to use laboratory equipments, perform basic tissue culture and molecular techniques. Read papers about the projects. Keep a detail notebook.They can work within a team. Learns the cells used in cell culture and makes research about them. Observes and analyzes the experiments and techniques. Learns the simple techniques that can be used in a cell experiment. Students will be able to research and plan selection and growth and maintanence of specific cell types. Protein analysis techniques are somewhat more complex. Therefore, some steps will be theoretical. In practical steps, the protocol should be followed exactly. For example; When gel electrophoresis, pouring the gel on a flat surface is very important for the wells to be smooth. Otherwise, proteins cannot be seen correctly. It is equally important to load the right amount of protein and dye. The student should also learn to apply cell culture protocols and some. For example; When passaging the cell, simply put 0.5ml trypsin into the t-25 flask. For trypsin inhibition, 1 ml of medium is enough. If you need to use a centrifuge device, 500 G will be enough for 5 minutes. Similarly, the day, date and who performed the transactions should be written on the flask. It is very important to work sterile and sterile pipette gun, serological pipette and centrifuge tubes.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Students will be informed about the basics of cell culture and the use of necessary materials and eqipment for this internship. These lessons will be given by the instructors and lab assistants in our laboratory. Each lesson will be given once and last for 3 to 4 hours.
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?
No spesific skills are required Student must have completed 1 year of medical study.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students
- Good Cell Culture Practice for stem cells and stem-cell-derived models.Pamies D; Bal-Price A; Simeonov A; Tagle D; Allen D; Gerhold D; Yin D; Pistollato F; Inutsuka T; Sullivan K; Stacey G; Salem H; Leist M; Daneshian M; Vemuri MC; McFarland R; Coecke S; Fitzpatrick SC; Lakshmipathy U; Mack A; Wang WB; Yamazaki D; Sekino Y; Kanda Y; Smirnova L; Hartung T. DOI:10.14573/altex.1607121
- Isolation; Culture; and Expansion of Mesenchymal Stem Cells.Ferrin I; Beloqui I; Zabaleta L; Salcedo JM; Trigueros C; Martin AG. DOI:10.1007/978-1-4939-6921-0_13
- Western blotting.Biji T.KurienaR. HalScofield https://doi.org/10.1016/j.ymeth.2005.11.007
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