##  INTRODUCTION

The laboratory mouse has emerged as the major mammalian model for studying
human genetic and multi-factorial diseases. Numerous mouse mutants have been
produced and, more recently, technological improvements have allowed mouse
mutants for virtually any gene to be produced by gene-specific approaches
(knock-outs, knock-ins and conditional mutagenesis). Random approaches such as
large scale, genome-wide ENU mutagenesis and gene trapping have also expanded
the current repertoire of available mutants. Using these mouse mutants,
researchers are able to decipher molecular disease and potentially develop new
diagnostic, prognostic and therapeutic approaches.  
  
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*To whom correspondence should be addressed. Tel: 44-1223-494451; Fax: 44-1223-494468; Email: damian@ebi.ac.uk 

The Author(s) 2009. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative
Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/ by-nc/2.5/uk/) which permits
unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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The International Knockout Mouse Consortium [IKMC
(http://www.knockoutmouse.org); (1,2)] is made up of four major projects
(EUCOMM (http://www .eucomm.org) in Europe, KOMP (http://www.nih
.gov/science/models/mouse/knockout/) and TIGM (http://www.tigm.org) in the USA
and NorCOMM (http://www.norcomm.org) in Canada, and is in the process of
producing mutations in ES cells for all known protein coding genes. A number
of mouse mutant lines have already been produced from these resources. In
particular, some 650 mouse lines are being produced and phenotyped in high-
throughput screens as part of the EUCOMM and EUMODIC projects (http://www

.eumodic.org), the results of which will be presented in the Europhenome
resource (3). To take this process to the next level, the International Mouse
Phenotyping Consortium (IMPC) has recently been formed with a remit to raise
the funding for and to coordinate the production of mouse mutants for each of
the IKMC mutations, along with high throughput phenotyping of these mice
resulting in the first complete catalogue of mammalian gene function (see
Appendix 6 of the PRIME final report: http://www.prime-eu.org/PRIME final
report.pdf).

Archiving and distribution of the products of these various projects is a
vital activity, alongside the capture of data describing in detail the
genotype and phenotype characteristics of the mutants. The costs for a typical
academic researcher to regenerate from scratch one of these knock-out (KO)
lines has been estimated at E25–30k and would take at least 9 months.
Regenerating the mouse lines is an obvious waste of public funds for science
as well as laboratory mice from an animal welfare aspect.

Since no single archiving facility can retain all of these mutant mouse
strains it is essential that all mutants that have been created are held in
centrally organised repositories, from which mutant mice can readily be made
available to interested investigators (4,5). The European Mouse Mutant Archive
[(EMMA); (6)] is a leading international network infrastructure for archiving
and provision of mouse mutant strains for the whole of Europe and worldwide.
To provide the best possible service to the international scientific community
there is a requirement for coordination of archiving and distribution of the
valuable genetically defined mice and ES cells in line with global research
demand. The Federation of International Mouse Resources [(FIMRe); (7)], of
which EMMA is a founding member and the European component, was initiated in
response to this need for coordination.

As well as coordination of archiving, there is a requirement for a common
portal that allows searching of all publicly available mice, including those
not from FIMRe partners, followed by redirection to individual repositories
for more detailed information and the possibility to order material. The
International Mouse Strain Resource [IMSR (http://www.findmice.org); (8)] has
been developed to fulfill this need and over the last few years, EMMA has
become one of the largest mouse network repositories worldwide and a major
contributor to IMSR.

EMMA also has a special role in the archiving and distribution of mouse
mutants as it is one of four repositories handling the mouse resources
produced by the IKMC initiative (EMMA archiving and distributing the mutant
mice arising out of the EUCOMM project, the KOMP repository
(http://www.komp.org) handling KOMP products, the Canadian Mouse Mutant

Repository [CMMR (http://www.cmmr.ca); (9)] handling the NorCOMM resources and
TIGM handling its own products. Eventually, these four resources will provide
access to data and material covering the complete, functional characterised,
proteome of the mouse, providing an unprecedented resource for bench
scientists studying all aspects of the mammalian genome including human
disease.

The EMMA resource database described in this paper provides up to date
information about the archiving status of mice and describes the genetic and
phenotypic properties of all the mutant strains that EMMA stocks. The EMMA
database has two main benefits to the research community: (i) scientists with
particular gene or genes of interest can discover if any mouse lines exist
with mutations in these gene(s) and what the observed phenotype changes were,
which may provide clues to the gene’s role, and (ii) it allows scientists to
order existing mouse mutants for further research and generation of data of
interest to other researchers. As well as providing user-friendly searching
and browsing of the database, the EMMA website is the link to the scientific
community and facilitates the submission of mice to the EMMA and requests of
mice from EMMA, as well as expressing interest in strains currently undergoing
archiving. The data recorded for each strain is a combination of data entered
by the original submitting scientist as well as subsequent curation to correct
and add extra value to the database. Although the full record is only
available through the EMMA database, summary data is exchanged with our
partners in IKMC and the IMSR to ensure that researchers using the portals
available at their sites see descriptions of EMMA lines, along with links back
to the original record in EMMA and the option to order biological material. In
addition, EMMA utilises the BioMart data management system (10,11) and the
Distributed Annotation System [DAS; (12)] to allow distributed, integrated
querying with other resources such as the Ensembl genome browser (13).

##  DATA COLLECTION AND CURATION

The EMMA website is used to advertise the goals of the project and encourage
interested parties to submit mouse mutant lines of widespread use to the
scientific research community as a disease model or other research tool. The
submission process is handled automatically by the website and collects
extensive data through a web form and stores this directly in the EMMA
database. Data collected at this stage includes:

. Contact details for the strain producer.

. Strain name, affected gene(s) and mutant allele(s).

. Genetic background of the original mutation and current background.

. Genetic and phenotype descriptions of the line.

. Bibliographic data on the line. . Whether the mouse models a human disease
and an OMIM ID if appropriate.

. Whether the strain is immunocompromised.

. Whether homozygous mice are viable and fertile and if homozygous mating are
required.

Additional optional data collected includes:

. Affected chromosome, dominance pattern and ES cell line(s) used for targeted
mutants.

. Name and description for chromosome anomaly lines. . Mutagen used for
induced mutant lines.

. Promoter, founder line number and plasmid/construct name(s) for transgenic
lines. . Breeding history of the line.

. Current health status of the line and specific information for animal
husbandry such as diet used.

. How to characterise the line by genotyping, phenotyping or other methods
e.g. coat colour.

. Research areas the mouse is useful for, and whether it is a research tool
such as a Cre-recombinase expressing line.

Extensive curation takes place to correct and augment the initial submission
data. To facilitate input of correct data by submitting users, specific tools
have been incorporated into the submission form, for searching and selecting
approved gene, allele, background names, symbols and identifiers (from the
Mouse Genome Database (MGD) developed by the Mouse Genome Informatics (MGI;
http://www.informatics.jax.org) group (14). Similar tools for searching and
selecting PubMed bibliographic references and identifiers have also been
implemented. However, there is still a requirement for manual correction of
submitted data using our curation interfaces.

The curation is based on the application of international rules and standards
for the initial assignment and periodic review and update of the strain and
mutation nomenclature, as defined by the International Committee on
Standardized Genetic Nomenclature for Mice
(http://www.informatics.jax.org/mgihome/nomen). These approved definitions
make use of control vocabularies for gene, allele, background names and
symbols. Specific automated routines and associated manual curation procedures
have been defined and implemented, in particular, for:

. Assigning to each submitted strain record a unique EMMA identification (ID)
as the primary attribute for internal strain identification and retrieval and
cross-reference with connected databases such as IMSR.

. Checking that the submitted records of mutant genes or expressed transgenes
(and corresponding alleles), carried by the deposited strains, have assigned
the correct names, symbols and identifiers, and mutation classification (as
defined by MGI) according to the associated bibliographic references.

. Proposing new mutant gene and allele names, symbols and identifiers for
publication in the MGD database, according to the associated bibliographic
references or personal communication with submitting scientists.

. Checking that the submitted backgrounds of deposited strains have approved
names and symbols assigned.

. Inserting a preliminary strain designation for each newly submitted strain,
including the assigned strain background name and the MGI allele symbol, and
associating it with the corresponding EMMA strain ID.

. Reviewing and approving the preliminary strain designations, in
collaboration with the curation group at IMSR.

. Periodically reviewing and updating of current strain designations,
according to variations of MGI gene and allele’s names and symbols.

. Automated correction and population of bibliographic data using the
submitted PubMed IDs and the CiteXplore web service (http://www.ebi.ac

.uk/citexplore/).

Archiving of submitted mice is handled by one of the EMMA mouse archiving
partners (CNR Instituto di Biologia Cellulare in Monterotondo, Italy; the CNRS
Centre de Distribution de Typage et d’Archivage Animale in Orleans, France;
the MRC Mammalian Genetics Unit in Harwell, UK; the Karolinska Institute in
Stockholm, Sweden; the Helmholtz Zentrum Mu¨ nchen in Munich, Germany; the
Wellcome Trust Sanger Institute in Hinxton; the Institut Clinique de la Souris
in Strasbourg and the CNB-CSIC, Centro Nacional de Biotecnologia in Madrid).
The archiving process involves genotype and/or phenotype verification of the
mouse, followed by test freezing of either sperm or embryos and then checking
the stock can be reconstituted from this frozen stock. Several strains are in
particularly high demand as they represent extremely interesting disease
models or valuable Cre-expressing lines and these are kept as live stocks
facilitating a fast delivery to the customers. The EMMA lines are supplied to
the research community for research purposes only and there is no charge for
the cryopreservation service. Archiving of mice produced by the EUCOMM mouse
production centres follows the same procedure except the initial import of
data describing these lines is automated from the EUCOMM database. The EMMA
database is used internally by the EMMA partners to track each mutant strain
through the archiving process. For example, the status of the strain in the
archiving pipeline, which centre is archiving the strain, the funding source
for this archiving, which material is currently in stock and available to
order is all stored in the database. EMMA archiving centres record this data
using internal interfaces implemented using Java Spring and Hibernate
technologies.

Requests for EMMA mice are also submitted via the EMMA website and recorded in
the EMMA database. The archiving centres again track the whole process of
distributing the requested mice using the database and the same internal Java
interfaces.

EMMA now contains over 1700 submitted strains from 19 countries including
around 50 lines from the USA, Canada and Australia. In the coming 5 years, it
is predicted that there will be a tripling of the mouse lines held, largely as
a result of the IKMC initiative. To date EMMA has sent out 1245 lines to
requesting scientists worldwide. Although nearly 58% of the requests for
mutant mouse lines were from European scientists, about one-third come from
the USA and Canada and requests from Asia are steadily increasing. So far,
EMMA has shipped mice to scientists from more than 500 different institutions
located in 39 countries. Considering the estimated cost of generating these
lines from scratch the existence of the EMMA resource has saved the worldwide
community E37M and 934 years of laboratory effort.

##  QUERYING THE EMMA DATABASE

The EMMA database can be searched using a userfriendly query interface (Figure
1). This search takes full/partial case-insensitive terms and searches against
the official MGI gene symbols e.g. Otog, the official IMSR designated strain
name e.g. B6.129S2-Otog  tm1Prs  / Orl, the common strain name e.g.
OtogC57BL/6J, the phenotype description e.g. auditory functions or EMMA IDs
e.g. EM:01820. EMMA lines are also browsable by the affected gene, mutant type
(e.g. Targeted Knock-out, Targeted Knock-in), particular research tools (e.g.
Cre-expressing lines) or mice produced by large projects (e.g. EUCOMM lines).
Results of searches or browsing are presented in a table, sortable by any of
the columns, which lists the EMMA ID, gene affected (with hyperlinks back to
MGI pages describing the particular gene and mutant alleles in detail), common
strain name, approved international name and a link to either order the line
or express interest in ordering lines that are in the process of being
archived. The latter option triggers an automated process, in which the
particular archiving centre is informed that there is a priority for this line
and when it becomes available further automated emails inform the original
scientist that they can go ahead and complete the ordering process.

Clicking on any of the strain names pops up a strain description (Figure 2)
including the mutation type, genetic background it is currently maintained on,
genetic and phenotype descriptions if known, the original producer, literature
references, the genotyping or phenotyping protocol needed to confirm the
mutation, what material is available along with delivery times and costs and a
link for downloading associated Material Transfer Agreement (MTA)
documentation, if applicable.

##  INTEGRATION WITH OTHER RESOURCES

As described earlier, a subset of data on each of the EMMA curated lines are
sent weekly to the IMSR, allowing users searching this common catalogue of
mutant lines to be redirected to our site for more detailed data and the
ability to order the line. The MGD database

Figure 1. Browsing and searching for mouse lines in EMMA. Relevant strains can
be identified by either (i) typing case-insensitive, full/partial terms in the
top text field which searches against the affected gene symbols and name,
approved international designated and common strain names, phenotype
description and EMMA ID, or (ii) browsing through a complete list of lines or
partial lists categorised by the affected gene(s), mutant type (targeted, gene
trap, transgenic, induced, chromosomal anomalies or spontaneous), research
tool [Cre recombinase expressing strains, lines for tetracycline
(Tet)-regulated gene expression systems], strains provided by the Wellcome
Trust Knockout Mouse Resource and finally strains produced out of the EUCOMM
programme. Results are presented as a table of the EMMA ID, affected gene,
common and approved international strain names alongside links to order or
register interest in ordering a line when it becomes available. Clicking on
any of the common strain names pops up a description of the strain.

Figure 2. EMMA strain descriptions. Data presented includes the mutation type,
genetic background it is currently maintained on, brief genetic and phenotype
descriptions if known, the original producer, literature references, the
genotyping protocol needed to confirm the mutation, what material is available
along with delivery times and costs and a link for downloading associated MTA
documentation, if applicable.

provides extensive descriptions of known mutant alleles and EMMA links to the
MGD pages, wherever possible as the definitive source for this data.

As well as our simple search box, we also provide an advanced BioMart query
interface, which is linked from the main search page (Figure 3). The BioMart
interface queries a denormalised snapshot of the EMMA database that is updated
nightly. Queries can involve complex combinations of query terms including the
affected gene symbols and MGI IDs, common and official strain names, EMMA IDs,
mutant type, original and maintained genetic backgrounds and the type of
material available (frozen embryos, sperm or ovaries, live mice on shelf or
mice rederived from frozen stock). The results are fully configurable,
allowing any combination of the fields presented in the standard EMMA search
results and strain descriptions to be displayed, as well as extra data such as
whether the mutant is viable and fertile when homozygous and whether it is
required to keep it homozygous, whether the line is immunocompromised, if it
represents a human model, the breeding history and for targeted mutants known
dominance and ES cell line used, and for transgenics the promoter and plasmid
construct used. The results can be previewed and exported in a number of
formats such as HTML, Tab/Commaseparated text or Excel. However, the real
benefit of BioMart comes from the ability to perform integrated querying with
BioMarts deployed on other resources, which share a common identifier such as
MGI or Ensembl IDs. For example, in Figure 3a BioMart query  
  
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Figure 3. The EMMA BioMart interface. This interface allows advanced querying
of the EMMA database as well as distributed and integrated querying with the
Ensembl resource. In this example EMMA targeted knock-out lines are identified
that have affected genes annotated by Ensembl as being located within the
first 100Mbp of chromosome 1 and containing a transmembrane domain in their
protein products. The results table is fully configurable from within the
interface and here shows the strain name, EMMA ID (hyperlinked back to the
strain description at EMMA), gene symbol and phenotype description from the
EMMA BioMart and the Ensembl Gene ID, chromosome, start and end from the
Ensembl BioMart

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located at the Wellcome Trust Sanger Institute, Hinxton, UK.

has identified all lines held in EMMA that have an affected gene annotated by
Ensembl as being located on the first 100Mbp of chromosome 1 and having a
transmembrane protein domain.

A new portal is currently being developed for the IKMC initiative by the
International-Data Coordination Center (I-DCC; http://www.i-dcc.org). This
will be released late 2009 and will display the status of all genes in the
mutagenesis pipeline along with available products and data for the mutant ES
cells and mouse lines. The portal will utilise a number of BioMarts developed
for the IKMC component mutagenesis pipelines and repositories, as well as for
other useful resources such as the GXD (15) and Eurexpress
(http://www.eurexpress.org) gene expression databases, and the Europhenome
phenotyping resource. The EMMA BioMart will form an integral component of this
IKMC portal and in addition allow a wider variety of integrated queries from
our EMMA BioMart server.

Another type of data integration is provided by our Distributed Annotation
System (DAS) server (www.emmanet.org/das). This serves up summary level data
for each EMMA line, allowing the display of EMMA strains on DAS clients such
as the Ensembl genome browser. For example by browsing to
http://www.ensembl.org/Mus_musculus/Gene/External
Data/EMMA?g=ENSMUSG00000055694 and clicking on the ‘Configure this page’
option and selecting EMMA it is possible to view any EMMA lines that exist for
this gene (Gdf1). The EMMA ID, affected gene symbol, name and link to curated
data at MGI is given along with the mutation type, phenotype summary and a
link to the strain description at EMMA.

##  CONCLUSION AND FUTURE DIRECTION

The number of mutant mouse lines submitted to EMMA as well as the number of
requests for these mutants is likely to increase significantly in the near
future. This is due to the large scale and systematic efforts of the IKMC to
perform saturation mutagenesis of the mouse genome using gene targeting and
gene trapping approaches. As well as continuing to expand the number of lines
curated and distributed by the EMMA resource, collaboration with international
efforts to present all available mutants worldwide is going to become ever
critical as the IKMC and eventually the IMPC initiatives continue to produce
and characterise mutants. Data exchange with IMSR will continue to provide a
common access site and EMMA will collaborate extensively with the I-DCC to
provide a central portal to the data and products produced by the IKMC. There
will be a particular focus on utilising the phenotyping data arising out of
these programmes to allow searching for mouse models using precise phenotype
queries structured using the Mammalian Phenotype (MP) ontology (16).

The EMMA project is currently funded until 2013, but obviously long term,
stable funding for the data storage and mouse archiving that EMMA performs
will be critical to capture and maintain the products emerging from the IKMC
and IMPC programmes. This is a recognised issue and the European Commission is
currently funding a number of projects under the ESFRI Roadmap with the goal
of identifying sources of long term funding for key scientific activities.
Infrafrontier (http://www .infrafrontier.eu) is one of these projects and is
tasked with securing such funding for archiving and phenotyping of mouse
mutants. Infrafrontier has already decided that the archiving aspect will be
taken care of by a major upgrade to the EMMA project. Hence, it is highly
likely that EMMA will continue providing this valuable service to the
worldwide scientific community for many years to come.

##  ACKNOWLEDGEMENTS

The authors would like to thank the members of the EMMA Technical Working
Group, Board of Participating Directors and EMMA archive centres who
coordinate and carry out the hard task of archiving all the mouse lines.

##  FUNDING

European Commission FP6 Infrastructure Programme [grant no. 506455]. Funding
for open access charge: European Commission FP7.

Conflict of interest statement. None declared.