Transgenic Mice and Genetargeting

Transcription

1 Transgenic Mice and Genetargeting mice

2 In Biomedical Science Techniques of transgenic and gene-targeting mice are indispensable for analyses of in vivo functions of particular genes and roles of their mutations in the development of human disease as well as genetic analyses of interaction of different genes.

3 Transgenic mouse To investigate the effects of overexpression of genes in particular organs Gene targeting mouse 1. Knock out mouse To investigate the physiological roles of genes during development 2. Knock-in mouse To investigate the effects of gene mutations found in diseases 3. Conditional knock out mouse To investigate the physiological roles of genes in particular organs at particular developmental stages

4 Design of Transgene Enhancer/Promoter cdna Poly A Tissue-specific enhancer/promoter Effect of overexpression of cdna in vivo Several copies~several hundred copies of transgene are tandemly integrated into chromosome

5 Advantage and disadvantage of transgenic mice Advantage 1. Technique is not complicated. 2. Transgene can be overexpressed in particular organs Disadvantage 1. Expression levels of transgene cannot be controlled 2. Integration site of the transgene is unpredictable

6 Mouse Embryonic Stem Cells (ES Cells) ES cells are established from inner cells of mouse blastcysts Culture conditions (including selection of serum) are critical to maintain the character of stemness

7 Generation of targeting vector Exogenous 5 homologous region Gene (neo) 3 homologous region Targeting vector DT-A Targeting gene Homologous recombination

8 Selection of positive ES clones by Southern blotting probe H WT Ret allele Targeting Construct exon11 D707 H exon12 9kb H H 9.0k WT allele 4.8k targeted allele D707N Targeted allele Ret D707N-neo H D707N 4.8kb H H

9 GDNF/RET signaling RET GDNF RET: receptor tyrosine kinase GFRα1 P P P P Neuronal survival / differentiation (eg. Enteric Nervous System) Kidney development (ureteric bud branching) Spermatogenesis

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11 RET Mutations in Human Diseases Disease Papillary Thyroid Carcinoma MEN 2A & MEN 2B Hirschsprung s Disease RET Mutations Rearrangement Cell (1990) Point Mutations (Nature 1993, 1994) Point Mutations Frame Shift, Deletion (Nature 1994)

12 Thyroid carcinoma Medullary carcinoma Papillary carcinoma

13 RET Rearrangement in Papillary Thyroid Carcinoma Sinal sequence Cysteine- Cadherin-relatedrich Kinase domain Human RET RI α H4 RET/PTC1 RET/PTC2 ELE1 RET/PTC3

14 Design of RET Transgene MMTV LTR RET cdna Poly A Mouse metallothionein Promoter/enhancer RET cdna Poly A

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18 Pigment cell tumor developed in RET-transgenic mice D B F

19 Clinical phenotype in Multiple Endocrine Neoplasia 2 MEN 2A MEN 2B FMTC Medullary Thyroid Carcinoma Pheochromocytoma Medullary Thyroid Carcinoma Pheochromocytoma Medullary Thyroid Carcinoma Parathyroid Hyperplasia Mucosal Neuroma Hyperganglionosis of the GI Tract Marfanoid Habitus

20 RET Mutations in MEN 2A, MEN 2B and FMTC Signal sequence Cysteine-rich Transmembrane Cadherin repeat Kinase domain RET Cys Cys Cys Cys Cys Cys (609) (611) (618) (620) (630) (634) Tyr Arg Trp Tyr Tyr Arg Arg Phe Tyr Ser Ser TyrSer Gly Gly Phe Trp MEN 2A & FMTC Glu Val Ser (768) (804) (891) Asp Leu Ala FMTC Ala Met (883) (918) Phe Thr MEN 2B

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22 Construct of RET-MEN2A gene for Transgenic Mice Cys 634 Arg MoMuLVLTR RET MEN2A Intron and polya #121 #180

23 Thyroid Tumor developed in MoMuLV/RET-MEN2A Transgenic mice wt tg

24 Medullary Thyroid Carcinoma developed in RET-MEN2A Transgenic Mice HE Staining Anti-calcitonin antibody

25 Serum Calcitonin Level in Transgenic Mice phenotype Bilateral MTC Unilateral MTC CCH Normal control No. animal Serum calcitonin level (pg/ml) 320± ±58 83±48 42±13

26 Physiological functions of RET RET Neural Development (Enteric Nervous System) Spermatogenesis Kidney Development

27 RET Mutations in Human Diseases Disease Papillary Thyroid Carcinoma MEN 2A & MEN 2B Hirschsprung s Disease RET Mutations Rearrangement Cell (1990) Point Mutations (Nature 1993, 1994) Point Mutations Frame Shift, Deletion (Nature 1994)

28 Hirschsprung s disease A common pediatric disorder with an incidence of 1/5000 births The congenital absence of enteric neurons in the distal region of the colon A marked intestinal dilation (megacolon) Hirschsprung s disease D707N mice absence of enteric neurons?

29 Short Segment Type(80%) Long Segment Type (12%)

30 RET Mutations in Hirschsprung s Disease Signal sequence Cysteine-rich Transmenbrane Kinase Domain RET P20L C142S S32L R231H L40P E251K P64L G93S R330Q F393L P399L R475Q C609Y C609W C618R C620R S690P E762Q S765P F873Q R972G F893L P973L S767R R897Q M980T K907E P1039Q L1061P E921K M1064

31 RAS RAF MEK1 ERK1/2 CREB ELK-1 SOS raft SNT/FRS2 GRB2 SOS GDNF GFR α 1 GRB2 RET py1062 SHC SHP2 Cell Survival Cell Proliferation GAB1/2 p85 AKT NF B κ p110 RHO FAK PI3-K RAC Lamellipodia Focal Adhesion Stress Fiber

32 Generation of RET Y1062F Knock in mice Wt RI RV RI 3 probe RI RV Targeting vector 8kb 5.5kb RV 19f Y1062F RV RI 19* PGK-neo 20 19r RET Y1062F KI RI RV 7.5kb * RV PGK-neo RI 20 RI RV Neo probe 4.5kb kb probe Neo probe c-ret Y1062F KI 270bp AAA CTC TAT GGCA K L Y AAA ATA TTT GGCA K I F : SspI restriction site Wt He Ho 140bp ret 9 ret 51 β-actin Wt Ho bp

33 Gross Appearance and Survival Rate of RET Y1062F Knock-in Mice A Ho Wt B survival rate % day after birth Wt (n=144) He (n=237) Ho (n=109) C body weight of KI mice g Wt He Ho P0.5 P13.5 P20.5 age

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35 D707N mice lack enteric neurons in the distal portion of the colon (2) Acetylcholinesterase staining (P3) D707N/D707N WT/WT

36 Normal Mouse Kidney RET Mutant Kidney

37 Findings of RET Y1062F Knock in Mice Kidney A k ub Wt u ad k u ub ad Ho B heart weight / body weight C kidney weight / body weight x x P0.5 P13.5 P20.5 Wt He Ho Wt He Ho P0.5 P13.5 P20.5

38 GDNF/RET signaling is essential for germ cell development Sperm GDNF Spermatogonial Stem Cells RET Sertoli Cells

39 Normal Testis Mutant Testis Hypoplasia of Testis in Y1062F mutant mouse Normal Testis Mutant Testis No sperm observed