Advanced Aminal Cell Culture

2013 2nd Semester

Department of Animal Science

Chungbuk National University

7th 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

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 KnockoutGene 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 Knockout

- 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 insertUsed for the Original Human Genome ProjectsIdeal for modification of Large fragments for the homologus recombinantion

Most of

Conditional Knockout

- Cre recombinase

- Why we need to make conditional knockout mouse ?

Embryonic Lethal

Oocyte Specific Knockout

- Cre recombinase are expressed in oocyte by ZP3 promoter- Desired locus has modified to have LoxP

Knock inTransgenic 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