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

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

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

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

Design criteria

Viewing designs Links to genome browsers Export for csv files

Viewing designs Design x

Viewing designs Conservation Repeats Design Gene Models mRNA transcripts

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

Viewing designs ConservationRepeats Design mRNA transcripts Gene Models

Assessing CE selection Transcript and Protein identifier links

Assessing CE selection Exons link

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

Assessing CE selection Coding Exons Protein Summary

KO: transcripts in VEGA

Fahd2a KO:Fahd2a Critical Exon

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

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

Custom design tool

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

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

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

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

Simple searches Simple searches using knockoutmouse.org

Simple searches Results page displays data from multiple resources

Simple searches Results page displays data from multiple resources

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

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

BioMart searches Select attributes for results output

BioMart searches Apply additional filters to further restrict results

BioMart searches Results from GWAScentral knockoutmouse.org

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

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

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

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

Resources Ensembl – VEGA – vega.sanger.ac.ukvega.sanger.ac.uk UCSC – genome.ucsc.edugenome.ucsc.edu MGI – IKMC portal – Europhenome – GWAScentral – BioMart –

Genbank Genbank files can be downloaded for every vector in IKMC

Genbank

Design criteria Max 3.5kb

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

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