bbc 2015

BeNeLux Bioinformatics Conference – Antwerp, December 7-8 2015
Abstract ID: P
Poster
10th Benelux Bioinformatics Conference bbc 2015
P26. PASSENGER MUTATIONS CONFOUND INTERPRETATION OF ALL
GENETICALLY MODIFIED CONGENIC MICE
Paco Hulpiau 1,2,3 *, Liesbet Martens 1,2,3 *, Yvan Saeys 1,2,3 , Peter Vandenabeele 1,2,4 & Tom Vanden Berghe 1,2 .
Inflammation Research Center, VIB, Ghent, Belgium 1 ; Department of Biomedical Molecular Biology, Ghent University,
Ghent, Belgium 2 ; Data Mining and Modelling for Biomedicine (DaMBi), Ghent, Belgium 3 ; Methusalem Program, Ghent
University, Belgium 4 . *paco.hulpiau@irc.vib-ugent.be, liesbet.martens@irc.vib-ugent.be
Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between
embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often
C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA
potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and
single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-
strain genome.
INTRODUCTION
Annotating the passenger mutations to the reported
genetically modified congenic mice that were generated
using 129-strain ESCs revealed that nearly all these mice
possess multiple passenger mutations potentially
influencing the phenotypic outcome. We illustrated this
phenotypic interference of 129-derived passenger
mutations with several case studies and developed a Me-
PaMuFind-It web tool to estimate the number and possible
effect of passenger mutations in transgenic mice of interest.
METHODS
We analyzed the SNP data release v3 from the Mouse
Genome Project available at Sanger Institute (Keane et al.,
2011). The data in the indel vcf file and SNP vcf file were
filtered to retrieve indels and SNPs present in at least one
of the three 129 strains (129P2/OlaH, 129S1/SvIm and
129S5SvEvB) and affecting the protein coding sequence
of the genes. These so-called protein coding variants are
based on the following sequence ontology (SO) terms:
stop gained, stop lost, inframe insertion, inframe deletion,
frameshift variant, splice donor variant, splice acceptor
variant, and coding sequence variant. In total, 949 indels
and 446 SNPs affecting 1,084 mouse genes were retained.
We gathered chromosome and gene start and end positions
for 1,084 genes covering 1,395 variations. The Ensembl
gene ID was used to find the most upstream and
downstream start and stop in all Ensembl transcripts for
that gene. Next these genome coordinates were used to
search for flanking genes within 2, 10, and 20 Mbps
upstream and downstream. We then downloaded all mouse
phenotypic allele data from the MGI resource and
extracted the data of genetically modified mouse lines.
Information on 5,322 genes (corresponding to 7,979 129-
derived genetically modified mouse lines) was connected
to genes with passenger mutations and affected genes.
Additionally we filtered the data to identify putative
regulatory variants. All data were stored in a MySQL
database and can be queried using the publicly available
web tool Me-PaMuFind-It:
http://me-pamufind-it.org/
Passenger genome mutations in gene-targeted mice (Nechanitzky and
Mak, 2015)
RESULTS & DISCUSSION
The vast majority of existing and well-characterized
genetically engineered congenic mice have been created
using 129 ESCs. 99.5% of these mouse lines are affected
by a median number of 20 passenger mutations within a
10 cM flanking region. This implies that nearly all
genetically modified congenic mice contain multiple
passenger mutations despite intensive backcrossing.
Consequently, the phenotypes observed in these mice
might be due to flanking passenger mutations rather than a
defect in the targeted gene (Vanden Berghe et al, 2015).
REFERENCES
Keane, T.M., Goodstadt, L., Danecek, P., White, M.A., Wong, K., Yalcin,
B., Heger, A., Agam, A., Slater, G., Goodson, M., et al. (2011).
Mouse genomic variation and its effect on phenotypes and gene
regulation. Nature 477, 289–294.
Nechanitzky R, Mak TW (2015). Passenger Mutations Identified in the
Blink of an Eye. Immunity 43(1), 9-11.
Vanden Berghe, T., Hulpiau, P., Martens, L. et al (2015). Passenger
Mutations Confound Interpretation of All Genetically Modified
Congenic Mice. Immunity 43(1), 200-9.
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