1. 7th Lecture
Advanced Aminal Cell Culture
2013 2nd Semester
Department of Animal Science
Chungbuk National University

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. 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. Knockout/Knock In/Transgenic
Animals

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. Knockout Mouse

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. Knockout mouse from Embryonic Stem Cell

9. Homologous Recombinantion

12. 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

13. Conditional Knockout
- Why we need to make conditional knockout mouse ?
Embryonic Lethal
- Cre recombinase

15. Oocyte Specific Knockout
- Cre recombinase are expressed in oocyte by ZP3 promoter
- Desired locus has modified to have LoxP

16. Knock in
Transgenic vs Knock-In?

17. Transgenic Animals
- Embryonic Stem Cell
- Somatic Nuclear Transfer
- Viral Vector

18. Novel strategy for generation of
knockout/knock-in/transgenic
- Zinc-Finger Nuclease
- TALEN
- CRISPER
Genome Engineering?

19. Zinc-Finger Nuclease

20. TALEN

21. CRISPR

22. One-step generation of Knockout
Animal using TALEN