7th Lecture

Advanced Aminal Cell Culture

2013 2nd Semester
Department of Animal Science
Chungbuk National University
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
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
Knockout/Knock In/Transgenic
Animals
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?
Knockout Mouse
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
Knockout mouse from Embryonic Stem Cell
Homologous Recombinantion
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
Conditional Knockout
- Why we need to make conditional knockout mouse ?
Embryonic Lethal

- Cre recombinase
Oocyte Specific Knockout

- Cre recombinase are expressed in oocyte by ZP3 promoter
- Desired locus has modified to have LoxP
Knock in
Transgenic vs Knock-In?
Transgenic Animals
- Embryonic Stem Cell

- Somatic Nuclear Transfer

- Viral Vector
Novel strategy for generation of
knockout/knock-in/transgenic

- Zinc-Finger Nuclease
- TALEN
- CRISPER

Genome Engineering?
Zinc-Finger Nuclease
TALEN
CRISPR
One-step generation of Knockout
Animal using TALEN

Cell culture 07

  • 1.
    7th Lecture Advanced AminalCell 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 CellCulture 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.
  • 5.
    Why we careabout 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.
  • 7.
    Knockdown vs Knockout GeneKnockout 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 fromEmbryonic Stem Cell
  • 9.
  • 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 - Whywe 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.
  • 17.
    Transgenic Animals - EmbryonicStem Cell - Somatic Nuclear Transfer - Viral Vector
  • 18.
    Novel strategy forgeneration of knockout/knock-in/transgenic - Zinc-Finger Nuclease - TALEN - CRISPER Genome Engineering?
  • 19.
  • 20.
  • 21.
  • 22.
    One-step generation ofKnockout Animal using TALEN