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Wellcome Trust Sanger Institute Team 87 High Throughput Conditional Mutagenesis KOMP EUCOMM ● Gene Trapping ● Gene Targeting [email protected] Conditional Allele lacZ-tagged allele (‘knockout first’*) FRT FRT βgeo 1 2 loxP 3 loxP Cre recombinase Flp recombinase FRT 1 2 3 1 FRT βgal 3 loxP conditional allele (wild-type) lacZ-tagged deletion allele Cre recombinase 1 3 deletion allele (∆ exon, frameshift, NMD) *based on Testa et al., Genesis (2004) Sanger Targeted Mutagenesis Program Intermediate Vector Targeting Vector ES cell ● Gene Annotation & Critical Exon selection - Havana ● Automatic Vector Design ● Gateway Adapted Intermediate Vector (from serial liquid recombineering) ● Final Conditional Targeting Vector (from 3-way Gateway reaction) ● ES Cell Production & Genotyping Gene Annotation & Critical Exon selection Havana ● Otterlace ● Phase shift – stop codon - NMD Automatic Vector Design LacZ-NEO_loxP loxP Critical coding exon 2 1 0 2 1 >0.1kb G5 0.3kb U5 0 0 0.1kb U3 0 phase >0.3kb D5 >0.7 kb 0 0 D3 G3 >0.7 kb ~1 kb ~5 (3) kb ~5 kb ● Candidate regions (U, D and G) at pre-defined distances ● 50'mer selected using Array Oligo Selector ● Unique in BAC Automatic Vector Design ● Public interface Gateway Adapted Intermediate Vector tet KanR pBAD Red RecA CE loxP loxP pSC101 (ts ori) CmR Fori CmTet Cm Cm Kan Tet Zeo/PheS R2 R1 R1 Heat induction cre C r e lPR cre cre R1 or cre pSC101 cre R1 Cm Z/P Cre Expressing E. Coli R1 R3 R2 R2 R1 R3 R4 R2 R3R4 KanR AmpR R2 Amp Kan Zeo R4 R3 purified plasmid DNA loxP ● R4 Amp Zeo Intermediate Targeting Plasmid ● R2 AmpR ColE1 ori R3 R4 Cm Kan ZeoTet linear pBR322 based plasmid serial liquid recombineering ● Indexed BACs ● Recombineering plasmid introduced ● 3 consecutive rounds of recombineering Resource Final Conditional Targeting Vector ori ClonNAT attL1 FRT SA 5’ arm βgal Targeting Element FRT l attL2 oxP neo pA attR1 attR2 3’ arm loxP 5’ homology arm Transform gyr+ E.coli critical exon B1 ZeoR PheS attR3 attR4 ori B3 Kan Chl-Phe AmpR CmR ori ccdB KanR βgal ori neo pA B4 KanR AsiSI Linearization site attL4 attL3 B2 SA PGK in vitro L/R clonase reaction DTA pA pA DTA PGK 3’ homology arm Final targeting vector Vector Backbone with Negative Selection ● 3-way Gateway reaction ● Modularity ● Resource ES Cell Production & Genotyping Pick colonies X-Gal staining 1/4 24 clones selected Genomic DNA 1/4 Master 1/5 Backup DNA plate Frozen archive ● Resource LR-PCR and sequencing QC LR PGK kan Intermediate Vector attR3 attR1 attR2 EM7 ZeoR attR4 PheS loxP Z1 R3F Z2 LRR R4R LR Targeting Vector attB3 attL1 FRT FRT βgal En2 SA neo attL2 attB4 pA loxP L3 L1 ES cell clone gf attL1 LAR3 R1R NF LRR RAF5 FRT βgal neo LR L4 gr attL2 FRT loxP En2 SA GF FCHK loxP loxP pA R2R LF LRR GR www.sanger.ac.uk/htgt Reporting by Gene, Vector or ES clones ● 3 reporting granularities – Gene – Vector – ES cell Reporting by Gene, Vector or ES clones ● 3 reporting granularities – Gene – Vector – ES cell Reporting by Gene, Vector or ES clones ● 3 reporting granularities – Gene – Vector – ES cell Reporting Detail ● Detail – Design – QC – synthetic constructs Reporting Detail ● Detail – Design – QC – synthetic constructs ● DAS for genome browsers Reporting Detail ● Detail – Design – QC – synthetic constructs Resource Reporting ● Resources – – – – Vega Annotated genes ● Queries and Links by: – Gateway-adapted Intermediate Vectors Final Conditional Targeting Vectors Targeted ES Cell Clones Gene ● Vega ● Ensembl ● MGI – Vector – ES clone BioMart ● www.sanger.ac.uk/htgt/martview ● demo.biomart.org – Thanks to Damien Smedley Portals ● www.sanger.ac.uk/htgt ● komp.sanger.ac.uk ● www.eucomm.org Contacts & Contributors ● Project Manager – Vivek Iyer ([email protected]) Project Coordinator – Wendy Yang – Darren Oakley – David Jackson ([email protected]) – ● Manousos Koutsourakis Genotyping & QC – ● Barry Rosen ● High-throughput vector construction – ● Irina Shurov ([email protected]) Vector Development – ● Informatics Bill Skarnes – ● ● Tony West ES Cell Culture – Wendy Bushell ● Sanger Mouse Informatics – Niels Adams – David Melvin Other Informatics Contributors – Tony Cox – Lucy Stebbings – Patrick Biggs – Jessica Severin
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