module 4: understanding ko designs
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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 - PowerPoint PPT PresentationTRANSCRIPT
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