1. Module 4: Understanding KO designs Mark Thomas Wellcome Trust Sanger Institute

2. Aims Understand design criteria and selection of critical exons View designs using genome browsers Assess the effect of a particular design on gene transcripts Search for design/allele information using BioMart

3. Design criteria One design, three alleles i)KO-first tagged allele ii)Wild-type conditional allele iii)Null-allele

4. Design criteria Tagged allele – requires insertion of reporter cassette, ideally located 5’ Wild-type conditional allele – requires native splicing to be maintained Null-allele – requires a critical exon that induces a frameshift Other considerations; - alternative splicing (ie. nAGxxnAG, exon skipping) - repeat elements - conserved elements - targeting efficiency (ie. Insertion size) - domain structure - translation reinitiation

5. Design criteria

6. Viewing designs Links to genome browsers Export for csv files

7. Viewing designs Design 213835 1.5x

8. Viewing designs Conservation Repeats Design 213835 Gene Models mRNA transcripts

9. Viewing designs Conservation Repeats Design 213835 mRNA transcripts Gene Models U-oligos D-oligos

10. Viewing designs ConservationRepeats Design 213835 mRNA transcripts Gene Models

11. Assessing CE selection Transcript and Protein identifier links

12. Assessing CE selection Exons link

13. Assessing CE selection Exon sizes (bp) Intron/Exon phases U-oligos (intron 2) D-oligos

14. Assessing CE selection Coding Exons Protein Summary

15. KO: transcripts in VEGA

16. Fahd2a KO:Fahd2a Critical Exon

17. KO: transcripts in VEGA Fahd2a KO:Fahd2a Exon 3 - frameshifted

18. KO: transcripts in VEGA Fahd2a KO:Fahd2a Exon 3 Exon 3 – Alt. frame Exon 1

19. Custom design tool http://www.sanger.ac.uk/htgt/design/designedit/show_create

20. Artificial intron designs AG|G or AG|A Knockout-first wt conditional Null allele

21. Artificial intron designs Exon 1 Exon 1b 1a Targeted allele

22. Using BioMart to search Simple searches using knockoutmouse.org More complex searches using biomart.org

23. Simple searches Simple searches using knockoutmouse.org Possible to browse by products, phenotype, location and name

24. Simple searches Simple searches using knockoutmouse.org

25. Simple searches Results page displays data from multiple resources

26. Simple searches Results page displays data from multiple resources

27. BioMart searches BioMart allows users to query databases using a standard interface.

28. BioMart searches GWAS Central - searching for interesting gene targets gwascentral.org Step 1. Use filters to restrict search parameters

29. BioMart searches Select attributes for results output

30. BioMart searches Apply additional filters to further restrict results

31. BioMart searches Results from GWAScentral knockoutmouse.org

32. BioMart searches knockoutmouse.org Vectors, ES cells and mice available

33. BioMart searches BioMart builds queries using a series of dropdown menus, identifying; Database selection Filters – defines search terms Attributes - selects results to be returned

34. BioMart searches Search results are returned in a table, containing links to data sources

35. BioMart searches Different databases/datasets can be linked in BioMart, allowing results to be combined. IKMC dataset Europhenome dataset

36. Resources Ensembl – www.ensembl.orgwww.ensembl.org VEGA – vega.sanger.ac.ukvega.sanger.ac.uk UCSC – genome.ucsc.edugenome.ucsc.edu MGI – www.informatics.jax.orgwww.informatics.jax.org IKMC portal – www.knockoutmouse.orgwww.knockoutmouse.org Europhenome – www.europhenome.orgwww.europhenome.org GWAScentral – www.gwascentral.orgwww.gwascentral.org BioMart – www.biomart.orgwww.biomart.org

37. Genbank Genbank files can be downloaded for every vector in IKMC

38. Genbank

39. Design criteria Max 3.5kb

40. Translation Re-initiation - critical exons should be as 5’ as possible Unless, the resulting KO translation is less than 35aa, as under these conditions translation may be reinitiated, producing an N-terminally truncated protein. Bcam

41. Translation Re-initiation ATGGAACCCCCTGACGCCCGCGCAGGGCTGCTGTGGCTCACCT TCCTGCTGTCGGGCTACTCAG GTTGATGGTACTGGGGCTCGACACCGTCTGGCTTCTGTGGAACCACAGGGCTCAGAGTTC CTGGGCACAGTCCACTCTCTGGGCCGCGTACCCCCATACGAGGTAGACTCTCGTGGGCGC CTGGTGATAGCAAAGGTCCAGGTGGGCGATGGACGGGACTACGTGTGCGTAGTGAAGGCT GGGGCAGCGGGTACCTCAGAGGCCACCTCAAGTGTCCGTGTGTTTG 1 MEPPDARAGL LWLTFLLSGY SG*WYWGSTP SGFCGTTGLR VPGHSPLSGP 51 RTPIRGRLSW APGDSKGPGG RWTGLRVRSE GWGSGYLRGH LKCPCV Translation