Cell culture 06
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Transcript of Cell culture 06
Advanced Aminal Cell Culture
2013 2nd Semester
Department of Animal Science
Chungbuk National University
6th Lecture
Syllabus
Date TopicsSeptember 5, 2013 Introduction : What is Cell Culture?
September 12, 2013 Cell Culture As Model System For Research
September 26, 2013 Cell Culture For Antibody / Protein Production
October 17, 2013 Protein Production/Purification
October 31, 2013 Stem Cell INovember 14, 2013 Stem Cell IINovember 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS
December 12, 2013 Final Exam
DateSeptember 26, 2013 Cell Culture For
Antibody / Protein Production
조유미 , Madhusumida
October 17, 2013 Protein Production/Purification 이미진 , 정용호
October 31, 2013 이영 , 윤준호November 14, 2013 Stem Cell I Jia Jia Lin, 염동현
November 28, 2013 Stem Cell II Zhao MingHui,권정우December 5, 2013 Transgenic Animals Lin Zili, 이상배December 12, 2013 Genome
Engineering/NGS 조유진 , 김상욱
Stem Cell II
Today’s topic : Reprogramming
Differentiated Cell vs Stem Cell
Cell Differentation
The process by which cells or parts of an organism become different from one and other and also from their previous state.
The process by which cells or tissues of an organism acquire the ability to perform their special functions.
How is this accomplished?
Selective gene transcription
In any given cell, only the genes necessary for basic metabolism
and that cell’s special functions are active.
* muscle cells - actin, myosin
* pancreatic acinar cells - digestive enzymes
* neurons – neurotransmitters
Mechnism of Cell Differentitation
How selective gene transcription could be achieved?
Messiner, Nature Biotechnol. 2010
Changes in epigenetic marks during Development
Bock et al., Mol.Cell 2012
Reprograming
How to return back to pluripotent state?
Things to do :
- Reset all of epigenetic marks which repress pluripotent factors maintenance genes(Activation)
- Reset all of epigenetic marks which activate lineage specific gene expression(Repression)
Natural Reprogramming : Sperm & Oocyte
Sperm is highly differentated Cell.After fertillization, DNA derived from sperm is demethylated rapidly
Smith et al., Nature 2012
Conclusion : Oocyte have ability to reprogramming terminally differentiated cell
Experiments by John Gurdon (1962)
Somatic Cell Nuclear Transfer (SCNT)
Animal Cloning
Stem Cell from Somatic Cell Nuclear Transfer (SCNT)
Drawbacks of Stem Cell Generation using SCNT
- Ethical Problems
• ES cell should be generated from Cloned Blastocyst• Human Oocyte is needed• Human Cloning?
- Technical Problems : Low efficiency
iPSC (induced Pluripotent Stem Cell)
‘Yamanaka Factor’
- Oct4, Sox2, Klf-4, c-Myc (OSKM)- Transcription Factors which express abundantly in Embryonic Stem Cell- Screened from 24 transcription factors expressed in ESC- Retroviral expression of these 4 genes in embryonic/Adult fibroblast transformCells into ‘Stem Cell Like’ cells
Molecular event of induced pluripotency
Cell Lineage Specific Gene
Pluripotency Maintenance genes
Limitation of iPSC
- Although iPSC is much similar with ESC, its properties is not exactly same with those of ESC
• Source of Original Cell• Condition used for the generation of iPSCDifferent epigenetic states Different tumorgenicity…
- Potential Tumorgencity of iPSC
- Retroviral vector : Danger in the tumor inducing
- Known oncogene (c-Myc) was one of component
- Low efficiency for reprogramming
- Less factors (Sometime Only Oct4 can induce pluripotency)
-Protein-mediated generation of iPSC
- Chemical mediated?
Efforts to overcome these Limitation
Methodologic Limitation
‘OSKM’ is not enough? Presence of ‘Break’
Rais et al., Nature 2013
Current Application of iPSC
- Research Tools : Patient specific cell derived from iPSC
ES Cell
ES Cell
High-throughput screening of drug candidate using iPSC derived cells