Cell culture 07

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Cell culture 07

  1. 1. 7th Lecture Advanced Aminal Cell Culture 2013 2nd Semester Department of Animal Science Chungbuk National University
  2. 2. Syllabus Date Topics September 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 I November 14, 2013 Stem Cell II November 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS December 12, 2013 Final Exam
  3. 3. Date September 26, 2013 Cell Culture For Antibody / Protein Production 조유미, Madhusumida October 17, 2013 Protein Production/Purification 이미진, 정용호 이영, 윤준호 October 31, 2013 November 14, 2013 Stem Cell I Jia Jia Lin, 염동현 November 28, 2013 Stem Cell II Zhao MingHui,권정우 December 5, 2013 Transgenic Animals Lin Zili, 이상배 December 12, 2013 Genome Engineering/NGS 조유진, 김상욱
  4. 4. Knockout/Knock In/Transgenic Animals
  5. 5. Why we care about that? - Knockout/Knock-In animal (mouse) : Disease Model For Medical Research - Immune-deficient Pig : Xenotransplantation? - Transgenic Animals : Animal Bioreactor? Production of useful protein in Milk or Egg? - Domestic Animal Breeding?
  6. 6. Knockout Mouse
  7. 7. Knockdown vs Knockout Gene Knockout Gene Knockdown - Expression level of mRNA was decreased by RNAi (siRNA/shRNA/dsRNA) - Gene itself are still intact - SilencingEfficiency is not ~100% - Sometime residual activities are sufficient for phenotypes - Relatively easy and fast to perform - You can do in embryonic lethal gene - Gene locus in chromosome is permanently removed/changed - Complete loss of genotype Difficulty & Time consuming Embryonic lethal gene : Conditional Knockout
  8. 8. Knockout mouse from Embryonic Stem Cell
  9. 9. Homologous Recombinantion
  10. 10. BAC recombineering BAC : Bacterial Artificial Chromosome Plasmid vector can have ~150kb of insert Used for the Original Human Genome Projects Ideal for modification of Large fragments for the homologus recombinantion Most of
  11. 11. Conditional Knockout - Why we need to make conditional knockout mouse ? Embryonic Lethal - Cre recombinase
  12. 12. Oocyte Specific Knockout - Cre recombinase are expressed in oocyte by ZP3 promoter - Desired locus has modified to have LoxP
  13. 13. Knock in Transgenic vs Knock-In?
  14. 14. Transgenic Animals - Embryonic Stem Cell - Somatic Nuclear Transfer - Viral Vector
  15. 15. Novel strategy for generation of knockout/knock-in/transgenic - Zinc-Finger Nuclease - TALEN - CRISPER Genome Engineering?
  16. 16. Zinc-Finger Nuclease
  17. 17. TALEN
  18. 18. CRISPR
  19. 19. One-step generation of Knockout Animal using TALEN

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