TRANSGENIC TECHNOLOGIES: Gene-targeting

Transcription

1 TRANSGENIC TECHNOLOGIES: Gene-targeting

2 Reverse Genetics Wild-type Bmp7 -/-

3 Forward Genetics Phenotype Gene or Mutations First Molecular Analysis Second Reverse Genetics Gene Phenotype or Molecular Analysis First Mutations Second

4 "Model" Organisms in Biology What allows us to use them? 1. All organisms share similar cellular machinery 2. All animals use this machinery in similar ways to direct embryonic development How about behaviors? -Species-specificity -Phonotypical homologues -Pharmacological homologues -Reverse genetic homologues

5 Mouse model of drowning?

6 Transgenic mice Transgenic mice Knockout mice Knockdown Gene-trap

7 Transgenic mice

8 Gene-knockout mice Early 1980, Mrio Capecchi: Homologous recombination in fibroblasts Martin Evans: Isolation of ES cells 1987, Mario Capecchi and Kirk Thomas First gene-targeting in ES cells 1989, Rudolf Jaenisch in MIT Generation of knockout mice, beta-2 microglobulin In Korea, 1993, H3 ES cell line 1995, IP3K, PLCb1 KO mice born

9 neo neo Transfection of targeting vector ES Cells into Blastocysts Chimera ES Cell F1 Germ-line F2 Embryo Transfer to Uterus Foster Germ-line Strain with recessive coat color

10 Introduce targeting vector into ES cells Timeline for the generation of ES cell-derived mice Identify homologous recombinants by DNA analysis Identify mouse Chimeras with high ES cell contribution Germline transmission Begin analysis Drug selection Colony growth and expansion Inject clones into blastocysts Sexual maturation of chimeras Identify male and female heterozygotes Sexual maturation of heterozygotes Identify homozygotes

11 EMBRYONIC STEM CELLS ADVANTAGE: -Totipotency -Manipulation in Culture (screening rare events) e.g. Lotto, 1/8 x 10 6 vs. homologous recombination10 6~8 USE: -Generation of Transgenic Mice -Gene Targeting-specific Gene Manipulation -Gene Trap-Random Gene Mutation

12 Early mouse development From Sedivy & Joyner Gene Targeting 1992

13 Derivation of Embryonic Stem Cells From ICM 1. Isolation of PND 3.5 blastocysts 2. Culture on embryonic fibroblasts (EF) 3. Isolation of hatched ICM 4. Trypsinization of ICM and culture on EF 5. Isolation of ES colonies 6. Karyotyping (40XY) 7. Characterization of germ-line competence From old ES cells 1. Plating of ~100 ES cells per 10cm dish 2. Cuture for 7-10 days with LIF 3. Isolation of single cell-derived colonies 4. Karyotyping (40XY) 5. Characterization of germ-line competence

14 ES or Embryonic stem cells: Blastocyst-stage cells that have been coaxed and coddled into growing in culture

15 Normal C57BL/6 Blastocyst (black) ICM ES cells 129/SvJae (Blackagouti) agouti black

16 Making chimera with Morula

17 A mouse with 3 parents

18 The first chimera and germ-line transmission in Korea!! FvB chimera Chimeras from foster mother Backcross with female having recessive coat color

19 Genes unable to be KO Multi-copy genes in the mouse genome Genes unexpressed in mouse Serotonin-N-acetyltransferase bp A1 a1g

20 Generation of targeting vector: Overall Procedure Isolation of cdna RT-PCR Isolation of genomic clones Phage library Bacterial Artificial Chromosome (BAC) Genomic PCR Restriction mapping of genomic clones Axon mapping Vector construction Insertion of neo cassette into the target exon Attachment of negative selection marker

21 Gene Targeting By Homologous Recombination X X Wild-type locus Targeting Vector X probe Neo TK Plasmid X sequence Targeted locus Neo probe 1. Homologous arm x 2 2. Neo marker 3. TK marker

22 Generation of targeting vector : Which exon should be targeted to make null mutation? Preferred Avoided - First coding exon - Functionally important exon - Exon can be skipped by alternative splicing - Number of nucleotides with a multiple of 3

23 Exon mapping Splice donor acceptor CCACATTgtn agCAGAA...CCACATTCAGAA......ProHisSerGlu...

24 Exon mapping CCACATTgtn agcagAA...CCA CAT TAA......Pro His STOP

25 Start Stop Start Stop Start Start Stop

26 neo Start Stop neo Start Stop neo Start Start Stop

27 Positive selection: ES cells that have the targeting vector: neo resistant Negative selection: Select against ES cells with random vector integration sensitive to anti-herpes drugs, FIAU, gancyclovir random Selected out 1 neo 3 HSVtk 1 neo 3 recombinant alive

28 Probe 1 Molecular screen: Eliminates random integrants without HSVtk Use flanking sequences 1 neo 3 Probe 2 targeted allele endogenous allele Probe 1 Probe 2 +/+ +/- random +/+ +/- random

29 Gene-targeting of a1g T-type channels in Mice 8.6kb Wild type locus B S E B EHH B S H H H B F1 B1 Targeting Vector B S E B E H Neo 12.6kb T K Disrupted locus B S E B EH Neo PGK B1 S H H H B (kb) /+ +/- -/- (kda) +/+ -/

30 Factors influencing targeting efficiency isogenic DNA (perfect homology) fold van Deursen J, Wieringa B. Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors. Nucleic Acids Res. 20: , size of region of homology exponential relationship Capecchi MR. Altering the genome by homologous recombination. Science. 244:1288-9, 1989 robust screen! Positive controls Run Repeat masker intrinsic features of the locus -Recombination hot spots

31 Modified KO technologies Knock-in Gene-trap Conditional KO -Region-specific -Time-specific

32 Knock-in technology for analysis of gene isotype function e.g. PLCbeta1 locus Start Stop C-DNA pa neo PLCbeta4 cdna

33 Gene traps Exon trap : Insertion into endogenous exon -Neo-pA Intron trap : Splice acceptor-ires-neo-pa Poly-A trap : Promotor-Neo-Splice donor

34 Gene traps are alternatives to knockouts promoter exons lacz pa Promoter neo R pa Splice acceptor Trapped gene (~random) Electroporate ES cells Select for neo R transcripts splicing identify trapped gene by 5 RACE, sequencing Select genes from gene trap library for blastocyst injection

35 You don t need to make gene-trap! International Gene Trap Consortium (gateway to all other databases) Manitoba Gene Trap Database Geoff Hicks Soriano Gene Trap Lines Omnibank (Lexicon) knockout clones - library genetics.com/omnibank/omnibank_ebiology.htm CMHD: Centre for modeling human disease, Mt. Sinai Hospital, Toronto, Canada Bay area resource of Mouse Mutations in Secreted and Membrane Proteins German Mouse Gene Trap Database

36 Conditional Ko by CRE-lox system GFAP promotor CRE glia Neuron e.g. Astrocyte specific KO

37 Conditional Ko by tet system CRE How to knockout a gene in adult stage by using tet-off (tetr) system?

38 Conditional Ko by CRE-lox system

39 Other Reverse Genetic Approaches Site-directed mutagenesis RNAi (sirna or shrna) Chemicals (Chemical Genetics)

40 Site-directed mutagenesis Point mutations, domain replacement

41 Gene Replacement

42 RNA Interference Method 1 Method 2 Method 3

43 Mechanism of RNAi

44 Forward and Reverse "Chemical Genetics"