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NIH Planning Meeting for a Knockout Mouse Project
Bethesda North Marriott
Rockville, Maryland
March 24-25, 2005
Chair: Geoff Duyk
Summary & Recommendations
Presentations at the workshop laid out the status of the current and planned
mouse knockout resources:
- There are currently about 10,000 null mutants (ES cells or mice) that have
been produced. Of these, there are slightly over 3200 targeted knockout mice
that have limited availability because they are not in public repositories
and therefore not readily available to the entire research community.
- The European Union Conditional Mouse Mutant Program (EUCOMM) plans to develop
a resource of 20,000 conditional mutations in a 129Sv ES cell background.
- C57BL/6 is the preferred mouse strain for use in research; therefore efforts
to establish more useful ES cell lines for generation of C57BL/6 knockouts
would be valuable.
- Existing repositories have the capacity and capability to handle storage
and distribution of additional resources.
Recommendations:
- The NIH's plan to construct a set of ES cells carrying null mutations with
reporters, as recommended by the Banbury meeting, is important. The addition
of 10,000 more nulls with a reporter will complete the resource so that there
is a null for each gene in the mouse genome. Such a resource would be complementary
to the set of conditional mutants that the EUCOMM project plans to generate.
- NIH should support continued efforts to develop the C57BL/6 ES cell lines
as a system for generation of knockout mutants in an experimentally tractable
genetic background.
- NIH should explore "repatriation" (collection) of existing ES
cells and mice to repositories as an alternative to remaking the null mutants.
However, issues such as quality control, relative expense, and potential IP
restrictions must be carefully evaluated before a decision is made one way
or the other.
- NIH should coordinate its efforts with other international programs that
are developing the comprehensive mouse knockout resource.
Introduction
In the fall of 2003, a meeting was convened at the Banbury Center of the Cold
Spring Harbor Laboratory to discuss construction of a public resource consisting
of a comprehensive collection of mouse knockouts, i.e. a null mutation in every
gene in the mouse genome. The meeting was international in scope and attended
by scientists from both the public and private sectors. The issues discussed
included the scope of the potential effort, type of mutations and the strain
background that would be most desirable, the feasibility of producing such a
collection in a cost-effective and highly efficient manner within a reasonable
period of time, the data management requirements, the dissemination of data
and materials, and the feasibility and costs of phenotyping the mutant mice
generated in the project.
The 2003 CSHL Banbury meeting recommended the value of a knockout mouse project
to generate a library of ES cells comprising a null allele, ideally conditional
null for each gene in the genome. However, because they doubted that existing
technologies were sufficiently robust to accomplish a genome-wide, high-throughput
effort to produce mice with a conditional null mutation, the meeting attendees
agreed that the project should use a mixed strategy employing both gene trapping
and gene targeting technologies to produce a comprehensive collection of mutant
ES cell lines representing null mutations for "all" mouse genes (~20,000)
in the strain 129 background. The resource would be used to produce live mice
(which would then be stored and distributed as frozen embryos and sperm). A
graduated scheme, represented as a pyramid with three additional tiers, was
recommended for the subsequent phenotyping phase of the project. Tier 1, or
basic, phenotyping would be done on nearly all of the mice created by the project
and the phenotyping would be done centrally. Tier 2 phenotyping would be carried
out for only a subset of mice, although transcriptome analysis would be done
centrally, and Tier 3 phenotyping would be specialized and performed by individual
labs. It was envisioned that the project would international in scope.
Since the Banbury meeting, there has been a significant amount of activity
around the world to organize mouse knockout efforts. Investigators in Europe
are planning the European Conditional Mouse Mutagenesis Program (EUCOMM) that
proposes to generate 20,000 mutations (12,000 conditional gene trap mutations
and 8,000 conditional targeted mutations) in ES cells. The group proposes to
archive and distribute the mutant ES cell lines centrally, along with existing
Cre recombinase-expressing transgenic mouse lines. Additionally, they will establish
300 mutant mouse lines from the conditional mutants produced as part of the
project and provide an online database for the dissemination of information
about the resources. EUCOMM is a consortium of eleven laboratories and is led
by groups at the GSF (German National Research Center for Environment and Health)
in Munich, Germany and the Sanger Institute in Hinxton, UK. Other mouse knockout
projects are under consideration in Canada, Australia, Japan and at the Sanger
Institute.
In addition to these newly planned focused efforts, there are, of course, a
large number of knockout mice that have been previously created in individual
research laboratories, both public and private, around the world in the course
of their specific research projects. The existence of these mice should be taken
into account in planning the development of the proposed comprehensive resource,
in order to avoid unnecessary redundancy. As a basis for doing this, the NIH
has begun systematically to develop information about existing knockouts. The
approach involved compiling data from the Mouse Genome Database (data curated
from the published scientific literature), and gene trap sequence data submitted
to the dbGSS section of GenBank, as well as conducting a new inventory of available
but unreported knockout mice. The latter was done through an e-mail solicitation
widely distributed to the research community. The results were collated by the
MGD.
The information obtained to date indicates that mutations have been constructed
in at least 8188 unique genes, by either gene-trap or targeted knockout methods.
The information-gathering effort also indicated that only a small fraction of
the mice or ES cells containing these constructs is publicly available and that
investigators were willing to make available another small, but significant,
number. One of the problems with this effort was that there was only a limited
response from investigators outside of the US. Therefore it is almost certain
that the current number is a significant underestimate of the number of unique
genes that have been already been knocked out by the research community.
Since the Banbury meeting, the NIH has also been considering its role in the
construction of a comprehensive mouse knockout and how to complement the efforts
of the international community. The NIH Planning Meeting for a Knockout Mouse
Project was organized to assess the status of research in the field eighteen
months after the Banbury meeting, to obtain updates on current projects and
to get input from the scientific community on a draft proposal for an NIH mouse
knockout program
Meeting Summary
The agenda for the NIH Planning Meeting for a Knockout Mouse Project was designed
to address a number of issues of importance to NIH planning effort:
- How many knockout mutations, and of what type, would be appropriate for
the NIH to contribute to a comprehensive international resource?
- What ES cell strain background is preferred by the mouse research community?
- What would be the optimal vector design? Should the knockouts be "simple"
null deletions marked with a reporter gene or should they be conditional mutations?
- What are the bioinformatics needs for the project?
- Is current technology adequate to produce a comprehensive collection in
a reasonable period of time at a reasonable cost, or is additional technology
development needed? Are there other technologies on the horizon that could
significantly reduce the value of a comprehensive set of knockout mutations
before the project could be completed?
- What are the repository needs for the project?
At the meeting, a series of panel discussions and break-out groups discussed
each of these issues. The following is a summary of those discussions.
ES Cell Background A panel of experts discussed the use of ES cells
derived from inbred lines, particularly 129/Sv and C57BL/6, and of a 129/BL6
F1 hybrid cell line. The F1 line is very to easy handle and produces a high
rate of germline mice. Despite the efficiency advantages, there was a strong
recommendation that issues of mixed background genetics were of overwhelming
importance. The influence of genetic mutation is most easily interpreted on
a defined background. As a gene is bred onto a different genetic background,
the role of modifier genes can be inferred. Moreover, a mixed genetic background
makes breeding of congenic lines difficult, especially if it is into to a third
genetic background. Thus the recommendation was to the use an ES cell line derived
from an inbred strain to allow breeding to pure genetic inbred when needed.
There remained the question of which inbred strain, 129/Sv or C57BL/6, should
be used. Strong reasons were presented for each alternative. In the end, a majority
of those present recommended the use of C57BL/6, as the preferred line of choice
for many groups including immunologists and neurobiologists.
There were several important caveats stated:
First, it was acknowledged that the attendees were not necessarily representative
of the mouse genetics community as a whole and the choice of the community might
be different. Second, it was also understood that there are still technical
issues in handling the BL6 system that need to be solved before it can be introduced
as the workhorse genetic background. For example, C57BL/6 ES cells are more
fragile and have not been tested for high throughput use, as is the case of
129 ES lines that have been used in gene trapping efforts. Cryopreservation
of sperm but not embryos is problematic for C57BL/6.Thus, there was also a strong
recommendation to support further technology development research on the BL6
ES cell system, e.g. to optimize culture conditions for ES cells and to develop
feeder independent BL/6 lines to reduce cost and increase efficiency.
Vector Design Given that the European and other efforts will be focused
on conditional mutations, the meeting recommended that the NIH should consider
providing a complementary resource of benchmark reporter-marked null deletions.
The "simple" deletions would provide an opportunity to generate baseline
data that could then provide the context for the interpretation of the data
from the more complicated conditional mutations, and the deletion/conditional
heterozygote would be a particularly valuable experimental reagent.
In designing an NIH program, the mutation-generation strategies should be fully
validated, have a benchmarked cost per allele, and result in mice with a high
germ-line transmission frequency. Any strategy employed must ensure that the
target vector resource remains useful in the future. The attendees also ratified
the Banbury meeting's conclusion that the mutations to be incorporated in the
comprehensive resource (whether existing or newly generated) must be fully validated
as to null/conditional/reporter status.
There was general agreement that the NIH should not undertake the creation
of a complete resource but, because of the large number of international efforts
already underway, the NIH effort must be cognizant of and complementary to the
already active efforts in Europe, the UK, Canada, and elsewhere. Thus, the attendees
strongly recommended that an NIH gene targeting-based program must be closely
coordinated with international efforts to ensure that appropriate choices of
genes to target are made. However, it was also considered crucial that there
be an agreement among all of the groups participating in an international mouse
knockout project that the resource generated be available to all users around
the world.
Finally, whatever strategy is chosen, BAC recombineering-ready libraries of
targeting vectors that can be reengineered for any future genetic alteration
of locus are a vital first-line resource.
Beyond the considerations of vector design and mutation strategy, it was acknowledged
that baseline gene expression and phenotype information would make the resource
more valuable. However, it was recognized that this would also increase the
cost significantly and, as a result, such characterization was not included
as a recommendation for the initial phase of the mouse knockout project at the
NIH.
Bioinformatics
The following information will be needed for a knockout mouse
project and accumulating the information will require bioinformatics solutions:
- Gene Model List: A canonical list of genes in the mouse genome, tied to
the accessioned genome and version number will be required for a targeting
strategy for the knockout project. Similarly, the results of the efforts to
knock out genes by trapping should be tracked using sequence from the knockout
event.
- Prioritization of Gene Models: Tools are needed to prioritize the gene models
to be targeted in the KOMP. This prioritization will need to be coordinated
with international efforts such as EUCOMM, IGTC and existing targeted knockouts.
Another decision that will have to be made will be whether to implement a
regional process (systematic by chromosome) of assignments or a functional
process (based on gene families, biological pathways or systems, or requests).
- Inventory of Existing Knockouts: The NIH's effort to catalogue existing
gene traps and targets is based on sequence data from the International Gene
trap Consortium deposited in dbGSS, published information from MGD, and an
inventory of unpublished knockouts. Further efforts are needed to get more
information from groups (in Europe, Japan, Canada and Australia) that were
not reached by the current inventory. If an effort were made to repatriate
individual lab-generated knockouts based on community agreed standards (see
below), informatics tracking tools will be needed to assess the cost-effectiveness
of that approach, as opposed to remaking the mutants.
- Tracking Database: Each knockout group will have its own laboratory management
database and it was agreed that a new, uniform lab management database for
all participating laboratories was not necessary. However, a single project
tracking center will be needed to track and consolidate project-wide results,
information about the status of assigned knockouts (allocation of gene regions,
vector and ES cell availability), and results (molecular event on graphical
browser, where to get the vector, cell lines, or mouse reagent resources).
This project tracking center will likely also need to point to the participating
laboratory database for further information and individual knockouts.
- Project-wide Quality Control Standards: The participating groups will need
to develop a set of common standards and then provide sufficient information
to characterize the molecular event from each knockout with high degree of
confidence (sequencing should be required).
- Annotation of the Mouse Genome: While this is not an explicit component
of the mouse knockout project, annotation of the mouse genome is an issue
of major interest to the scientific community. The Sanger's VEGA annotation
project is interested in providing detailed annotation of the genome, but
is not currently funded for the entire mouse genome.
Technology Development
The issue of evolving technologies that might
obviate the need for a mouse knockout resource within the next few years was
discussed. It was agreed that siRNA-based technologies for selectively knocking
out gene function are likely to be a good complement to genetic knockout techniques,
but are not going to replace them. Additionally, there are no conditional transcription
technologies robust enough at present to compete with the recombinase-mediated
conditional approaches utilized in mouse knockout systems. There are new inducible
technologies that may be useful but at present the data are too preliminary
to assess as they have not been validated in mice. Additionally, many of these
usually include a transgenic system expressed with a Knockout. Thus the KO resource
is still a valued resource.
The group agreed that the availability of primary targeting vectors would be
extremely useful in facilitating the secondary modifications of alleles. There
is a need to engineer additional mouse strains to support conditional strategies
and no need to preconfigure ES cells for this effort. There are a variety of
histochemical and fluorescent protein markers that can be expressed in transgenics
or knock-ins and are available to assess the activity of integrases and recombinases,
and the group felt that technology is moving rapidly and many new tools are
appearing. There was consensus that the ideal reporter or recombinase expression
system has not been validated. There was no strong feeling among the participants
that any new approach is ready to be incorporated into mouse knockout projects
or that there is a limitation of availability of existing tools.
To make the conditional knockout resource maximally useful, the loxP/Cre-based
approaches are favored, but data presented at the meeting raised a concern about
secondary chromosomal damage as a consequence of Cre activity. Some preliminary
sites suggested that Cre induction can induce deletions that can be as large
at 20Mbases, presumably due to recombination between cryptic loxP sites in the
mouse genome. If the Cre-lox system is to be used, good Cre driver strains are
critical to the maximal usefulness of a conditional resource, and this is an
element of the system in which the private sector has a particular interest.
However, no proposal for developing a specific set of strains was discussed
at the meeting.
Repositories As a public resource, it will be essential for public repositories
to handle the products produced by the knockout mouse project. The following
points were made in the discussion of this issue:
- Repository efforts should be centralized. This will improve efficiency and
ensure the quality of the materials distributed to the user community.
- New repositories are not needed to handle the products of a knockout mouse
project; existing repositories have the capacity to scale up to accommodate
the output of the proposed project.
" The repositories must have expertise in handling, storing and distributing
ES cells, embryos, sperm and mice and must have good standards for quality
control for each type of product.
- The repositories must also have capability to create mice from ES cells.
- The repositories will need to provide user access to all information on
the strains they maintain.
- There are currently bottlenecks in distribution that need to be addressed.
- Users will need to share the cost of access to materials in the repository.
- Repositories will need to put in place plans to replenish, expand and extend
the knockout project's materials in the repository.
- Repositories will need to Integrate with other international resources (the
International Federation of Mouse Repositories, the Mouse Genome Database,
The International Mouse Strain Repository, the National Center for Biotechnology
Information, Ensembl, and the University of California, Santa Cruz).
Feedback to the NIH
Considerable progress in producing knockout constructs and mice has been made
since Banbury. The NIH's plan to construct a set of ES cells carrying null mutations
with reporters, as recommended by the Banbury meeting, is important. The addition
of 10,000 more nulls with a reporter will complete the resource so that there
is a null for each gene in the mouse genome. Such a resource would be complementary
to the set of conditional mutants that the EUCOMM project plans to generate.
However, there is considerable interest in continued efforts to develop the
C57BL/6 ES cell lines as a system for generation of knockout mutants in an experimentally
tractable genetic background. Private sector scientists are interested in "humanized"
mice, that is mice in which a human gene has been "knocked in" in
to replace the corresponding mouse gene. They urged NIH to consider this element
in its planning.
While generation of new mutants to complete the knockout resource is important,
participants urged NIH to explore "repatriation" (collection) of existing
ES cells and mice to repositories as an alternative to remaking the null mutants.
It was noted that there are, issues such as quality control, relative expense,
and potential IP restrictions that must be carefully evaluated before a decision
is made one way or the other. Overall, there was agreement that whatever initiative
NIH puts forward, it will need to coordinate its efforts with other international
programs that are also participating in developing the comprehensive mouse knockout
resource.
Finally, a warning that a complete catalog of mouse knockouts will totally
alter the sociology of the mouse research community landscape. In that regard,
it will be a disruptive innovation, and will create the need for an extensive
educational/re-training platform. Such education programs will facilitate the
wide-spread distribution of mice, advance research, and minimize duplicative
efforts.
Last Reviewed: August 26, 2008
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