Module 4: Understanding KO designs

Module 4: Understanding KO designs

Mark ThomasWellcome 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

Design213835

1.5x

Viewing designs

Conservation

Repeats

Design213835

Gene Models

mRNA transcripts

Viewing designsConservation

Repeats

Design213835

mRNA transcripts

Gene Models

U-oligosD-oligos

Viewing designsConservation

Repeats

Design213835

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 selectionCodingExons

Protein Summary

KO: transcripts in VEGA

KO: transcripts in VEGA

Fahd2a

KO:Fahd2a

Critical Exon

KO: transcripts in VEGAFahd2a

KO:Fahd2aExon 3 -

frameshifted

KO: transcripts in VEGA

Fahd2a

KO:Fahd2a

Exon 3

Exon 3 – Alt. frame

Exon 1

Custom design tool

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

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 searchSimple searches using knockoutmouse.org

More complex searches using biomart.org

Simple searchesSimple searches using knockoutmouse.org

Possible to browse by products, phenotype, location and name

Simple searchesSimple searches using knockoutmouse.org

Simple searchesResults page displays data from multiple resources

Simple searchesResults page displays data from multiple resources

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

BioMart searchesGWAS 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 searchesBioMart builds queries using a series of dropdown menus, identifying;

• Database selection• Filters – defines search terms• Attributes - selects results to be returned

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

BioMart searchesDifferent databases/datasets can be linked in BioMart, allowing results to be combined.

IKMCdataset

Europhenomedataset

ResourcesEnsembl – www.ensembl.orgVEGA – vega.sanger.ac.ukUCSC – genome.ucsc.eduMGI – www.informatics.jax.org

IKMC portal – www.knockoutmouse.orgEurophenome – www.europhenome.orgGWAScentral – www.gwascentral.orgBioMart – www.biomart.org

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

ATGGAACCCCCTGACGCCCGCGCAGGGCTGCTGTGGCTCACCTTCCTGCTGTCGGGCTACTCAG

GTTGATGGTACTGGGGCTCGACACCGTCTGGCTTCTGTGGAACCACAGGGCTCAGAGTTCCTGGGCACAGTCCACTCTCTGGGCCGCGTACCCCCATACGAGGTAGACTCTCGTGGGCGCCTGGTGATAGCAAAGGTCCAGGTGGGCGATGGACGGGACTACGTGTGCGTAGTGAAGGCTGGGGCAGCGGGTACCTCAGAGGCCACCTCAAGTGTCCGTGTGTTTG

1 MEPPDARAGL LWLTFLLSGY SG*WYWGSTP SGFCGTTGLR VPGHSPLSGP 51 RTPIRGRLSW APGDSKGPGG RWTGLRVRSE GWGSGYLRGH LKCPCV

Translation