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Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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Mice Traveling the World: Issues in Sharing and Transferring Mice

Lili M. Portilla

This presentation focuses on some legal issues having to do with mice transfers. I am not a lawyer, but I have associated with lawyers throughout my career. My disclaimer is that the views and opinions I am expressing are those of NIH. If there is any legal question, my advice is that you seek legal advice from your institution.

I will first set the stage on how NIH approaches the sharing of animal resources, data, and other things. A lesson learned at NIH is that we do not patent research tools. When this happens, the flow of these tools to researchers is restricted and academic research is hindered. Hence NIH’s position, like that of most academic centers in the United States, is that these research tools will not be patented.

However, if industry requests some of these research tools, there is nothing to prohibit us from licensing them, even though they are unpatented, and we can still realize some profit if they are used for commercial purposes. So patenting in and of itself is not the only way of guaranteeing a royalty stream for your institution.

The basic precept at NIH is that we expect our funded researchers, as well as our internal researchers, to make resources developed under our grants available to the research community and as unencumbered as possible. The NIH model organism-sharing policy covers all projects that may produce model organisms with the intent that they will be made available to the research community.

In 2003 NIH produced a new guide notice, that grant applications of $500,000 or more of direct costs in any single year are expected to include a plan on data sharing, meaning that the research institution and the researcher have to demonstrate to the NIH how they are going to make these resources available, be it through a material transfer agreement (MTA) or deposit in a repository. A general consensus is that this position promotes good citizenship in the life sciences community.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

As a matter of policy at NIH and many academic institutions, the technology transfer offices ask for documentation of data sharing using an MTA. This agreement specifies how these resources can be used and limits the transfer to a third party, and thus prohibits transfers to other institutions. Also it puts the requester on notice that they have to attribute the donor, the person that gave them the resource, in a publication. Without an MTA it is not clear to NIH whether investigators get proper attribution on transferred materials.

We at the NIH found that the existing agreements did not address the uniqueness of animal models and crossbreeding issues. Therefore, we developed our specific form to transfer animals called the “Material Transfer Agreement to Transfer Organisms.” It is a modified NIH standard agreement, but contains special terms. First, it specifically defines the allele. From an intellectual property perspective, this criterion is of most value in these resources so it is necessary to clearly identify the special allele or knockout; for example, the form would define a Brca1 floxed mouse (Brca1 floxed allele expressed in a mouse). The agreement may be used for any animal model. The information in the form also defines what is included in the material—for example, unmodified derivatives and unmodified progeny, zygotes, embryos, and cells, tissues, etc.

There is also language that allows for crossbreeding. This language is primarily for nonacademics, so they know NIH allows it and if you plan to distribute this crossbreed, please let the recipient know that this original allele was obtained by the NIH.

The agreement also contains addenda that address some of the intellectual property issues that come up with mice like Cre-lox and OncoMouse. There is also an animal transfer addendum; the form is available online (www.ott.nih.gov/forms_model_agreements/).

Finally, if you think your mouse incorporates third-party intellectual property, it is best to consult with your technology transfer office to determine how best to deal with this. In most cases, transfers between academics incorporating third-party intellectual property are not a problem. The problem and the sticking point come when these transfers are done for commercial purposes, in which case it is best to get some advice on how to proceed. In addition, check to see if your institution already has a license agreement in place, because this may facilitate the transfer. Sometimes these license agreements define how you can further transfer these mice—for instance, research purposes only and to academic institutions, no transfer to commercial entities. Your tech transfer office or your legal office can help you if there is an existing agreement at your institution.

Following are some questions that are posed frequently about animal transfers:

Can I transfer a mouse that I receive from my colleague at institution A to another colleague at institution B? Most of the time, the answer is no. If you signed an MTA, in most cases it says that you cannot transfer to a third party. In these cases we always go back to the original person that gave us the mouse or

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

the intellectual property and make sure it is okay to transfer it to another individual. In most cases it is not an issue, but it should not be done if the agreement you signed prohibits it.

Can I crossbreed a mouse developed in my lab with one that I received from my colleague in another institution? Proceed carefully. Usually, we make sure that crossbreeding language was put into the agreements if that was what the investigator intended. But many times people sign agreements containing a statement that you can’t crossbreed. My advice is to read what you sign. If you want to modify it, go back to that institution to say that you want to modify the agreement

Why is the institution asking me to sign both a material transfer agreement and an animal transfer agreement? The animal transfer agreement is very different from a material transfer agreement. The MTA specifically deals with intellectual property issues, whereas the animal transfer agreement deals with the care and use of a particular animal and is usually signed off by the vet in the institution. When these two documents are put together, they cause confusion. So in the new agreement that I just discussed, all these terms have been incorporated in order to avoid having multiple signatures and too much paper. They are different and they do serve different purposes.

Is it okay for me to deposit mice that I developed in my lab in a public repository? My answer is a big yes, but make sure that there is not any kind of intellectual property issue that would prohibit you from doing that. It may be necessary to consult with your legal and tech transfer people to make sure that’s not going to be an issue. From the perspective of NCRR, depositing an animal is ideal and removes the financial and resource burden from the lab of having to ship mice. We encourage our grantees that develop resources through grants to deposit them in many of the NIH-funded repositories.

A colleague requesting my mouse wants me to ship it to their animal contractor. Is this allowable? The answer is: it depends. For example, a particular institution used a contracting facility, like Charles River, to clean their mice and cage their mice. Once the mice were ready for the experiment to start, they would be shipped to another location. We thought that was fine, because that contractor was acting as an agent for the institution. But the issue comes in if the contractor is collaborating with the institution, in which case they almost become a third party in the transaction. It may become necessary to ask questions or have your tech transfer people ask some questions on how to proceed. Most of the time this is not an issue, as long as this relationship with the contractor is one of an agent with the institution.

Some helpful hints have arisen over the years. One is to keep your tech transfer office or legal office informed of what you are going to be doing with these mice. Tell them ahead of time if you plan on crossbreeding the mice or sharing them with another institution. It is better to address these terms at the beginning of the negotiations as opposed to after an agreement has been exe-

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

cuted, which is always more difficult and time consuming. So tell us as much as possible upfront. Another issue is defining the timeline for experiments. In our office, if we knew that an investigator had something time sensitive coming up, if they had to time it right with the animal shipping folks, the forms would get bumped up in priority in order to get processed, because the last thing we wanted to hear was that the mice were past their prime for the experiment and the investigators were not able to do anything.

When publishing results, if a paper is coming out describing a new knockout, it is useful to presign agreements with the mouse model on them so that when the investigator gets requests, s/he only needs to sign the agreement and ship the mouse off. As noted earlier, NIH encourages our investigators and our grantees to deposit their mice in repositories, to save time, effort, and money in their labs.

Another issue deals with shipping and timing. It is not advisable to ship mice in the heat of the summer. It is helpful to work with the shipping staff to make sure that the agreement is done so they do not need to wait for the paperwork to ship mice.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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Knockout Mouse Databases: The Knockout Mouse Project and Repository

Franziska Grieder

The mouse has played a key role in many discoveries and advances related to biomedicine and has contributed to improvements in human health. In part, this is because mice offer the following advantages (in addition to their small size and relatively short reproductive cycle): mice are well characterized (e.g., their entire genome has been sequenced), they are genetically similar to humans, and they exist as inbred lines and strains. Further, these strains can carry different mutations that mimic pathologic or disease conditions seen in humans.

In the past, spontaneous genetic mutations in mice have contributed to understanding the biology of human disease in significant ways, but today’s focus has shifted to induced, genetically engineered, or modified mouse strains. Specifically, the ascent of new powerful genetic methodologies applied to molecular biology has allowed scientists to delete or insert genes. Attention has shifted from the original inbred strains developed by Castle and Lathrop almost a century ago to technologies that produce tissue- and disease-specific biomedical models. These transgenic technologies have led to the creation of “knockout” animals, using targeted mutagenesis or gene replacement approaches that allow scientists to inactivate single genes by replacing or disrupting them with the introduction of exogenous DNA constructs. Seminal to this revolutionary development were studies conducted by three scientists who received the 2007 Nobel Prize in Medicine or Physiology for their achievements: Drs. Mario Capecchi, Oliver Smithies, and Martin Evans.

Recognizing the power of knockout mouse technology and its general widespread benefit to biomedical scientists, a group of scientists and experts from around the world assembled in 2003 at the Banbury Conference Center at Cold Spring Harbor Laboratory to explore the feasibility of creating a comprehensive, genomewide, and publicly available knockout mouse resource. This effort would generate a library of mutant mouse embryonic stem cells (ESCs) containing knockout or null mutations in every protein-coding gene in the mouse genome. The driving force behind this effort was the recognition that only a small fraction

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

of the approximately 20,000 to 22,000 mouse genes had already been knocked out and published, and most of these knockout mouse models were neither readily available nor accessible to the wider research community.

The creation of a comprehensive, widely available, and standardized (e.g., mouse strain background, genotype testing, and specific pathogen–free) knockout mouse resource in a timely and cost-effective manner would require a highly coordinated effort among several international partners. Thus the foundation of several knockout mouse projects was born.

Three independent but collaborative efforts have been launched to address the original challenge posed during the Banbury Conference: the Knockout Mouse Project (KOMP) funded by the National Institutes of Health (NIH) in the United States; the North American Conditional Mouse Mutagenesis (NorCOMM) Project funded by Genome Canada and its partners; and the European Conditional Mouse Mutagenesis (EUCOMM) Program funded by the European Union. I will focus on KOMP.

In a collaborative effort among different NIH institutes and centers, a five-year and greater than $50 million mutant mouse resource initiative was started in 2006 that aims to (1) use gene targeting to make a resource of null alleles marked with reporters, (2) support a repository to archive and distribute the products of this resource, (3) develop improved and robust ESCs on the inbred mouse strain C57BL/6, and (4) implement a data coordinating center that allows all scientists easy access to data relevant to this effort.

The KOMP Repository (www.komp.org) activities were awarded to the University of California, Davis (UC Davis) and Children’s Hospital Oakland Research Institute (CHORI) in Oakland, CA. They will, in a collaborative effort, be responsible for the preservation, protection, and distribution of about 8,500 knockout mice and related products for use by the research community. Additionally, the repository provides expert advice and assistance for scientists with all questions related to mouse biology and reproduction, cell culture techniques, molecular biology, and insight into the selection of the most appropriate model or approach for their research project.

The specific activities of the KOMP Repository include acquiring vectors, ESCs, and mice from the KOMP production teams. Next, all ESCs received are archived and expanded for quality control testing, including viability-growth-morphology and pathogen assessment. Prior to release for distribution, a percentage of clones undergoes genotype verification and chromosome counting. The repository also performs in vivo testing, which includes microinjection to produce high-percentage chimeras and germline transmission testing. Any generated products as a result of the testing (e.g., germline-transmitted mice, embryos, and sperm) are archived as well.

The repository allows the customer to learn about KOMP and its operations, and also provides access to the KOMP catalogue, to order products and express interest in and nominate genes that will be targeted with higher priority by the production teams. The KOMP website allows researchers to obtain help

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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(e.g., protocols for genotyping, microinjection) or information on issues related to material transfer agreements.

During KOMP’s first two years of operation, numerous joint meetings among the knockout mouse production programs have been held on an international level (i.e., KOMP, NorCOMM, and EUCOMM). Participants work hard to establish collaborations and coordinate activities in order to avoid duplication of efforts. The projects in North America and Europe have agreed to share their gene lists and data in order to help with the coordination. Ideally, resources produced by one project would be available to scientists on a different continent, thereby enabling scientists to simply order all mouse strains locally, thus avoiding the hassle of international transport. The future will tell if this becomes a reality and if sharing of mutant mouse resources can happen across international borders.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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NorCOMM, the North American Conditional Mouse Mutagenesis Project

Colin McKerlie

The focus of this presentation is NorCOMM, the North American Conditional Mouse Mutagenesis Project, in particular where it came from and the contribution of my laboratory, which is the archive and distribution program. In 2003, some collaborators and I at Mount Sinai Hospital Samuel Lunenfeld Research Institute were engaged in a genomewide random mutagenesis project using ethylnitrosourea, or ENU, to produce point mutations randomly across the mouse genome. We then applied a very comprehensive set of screens or phenotyping to try to identify the expression of those mutated genes by looking for abnormal phenotypes.

My contribution was from my pathology phenotyping lab, where we were using gross, histo-, and molecular pathology techniques to try to identify novel mutations or the expression of those mutations as pathology phenotypes. We were also using our pathology techniques to characterize phenotypes in unusual or abnormal mice screened out by my colleagues.

As pathologists tend to do, we collected and kept a lot of information and were, by necessity, in large part establishing a repository. It was a necessity because in a dominant screen that we were using for ENU, we were often analyzing, often at necropsy, uniquely mutagenized genomes. If we needed to go back and study a particular mouse because we were interested in that mutation or phenotype, we needed the ability to go back to the freezer, recreate that mouse, and put it through the process. In essence, then, we established a very comprehensive archive, starting with tissue, sperm and ovaries, and, of course, embryos.

Recognizing the increasing demand across Canada among investigators using the mouse as a model system, we took the opportunity to start archiving and distributing their lines, driven by two requirements. One was for ease of distribution, to take some of the burden from these individual investigators and centralize the resource, making distribution more efficient, and provide an ad-

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

vantage in terms of protection from a disaster as well as some additional opportunities. Thus, we established the Canadian Mouse Mutant Repository (CMMR).

New space was built for this; we have just relocated fairly recently into the Toronto Centre for Phenogenomics (TCP). Space was designed for receiving mice and allowing us to freeze them in various formats in liquid nitrogen or mechanical freezing—somatic tissue, embryos, sperm, ovaries, etc. The facility also allows us to go back into our repository to restore a mouse line. Large and secure capacity is an essential part of our repository and we also have redundant capacity to guard against catastrophic loss. In summary, the TCP, a large mouse-based research-enabling center with very large capacity (36,000 mouse cages), offers a very comprehensive set of services to enable the collective research enterprise to archive and distribute their mice efficiently.

The CMMR was established to focus on requirements of investigators prior to the more recently emerging International Knockout Mouse Consortium (IKMC). The CMMR website showcases all the mutants and our samples. All our lines are catalogued through the international mouse strain resource, hosted by the Jackson Laboratory (JAX). The importance of this repository is not only to make lines available and visible but also to make them accessible, although accessibility does not necessarily always correlate with usability. Our services are also identified, including embryo services, ovary, sperm, or somatic tissue.

The IKMC is an attempt to essentially have a single Web portal for the mouse-using investigator community to find a mouse that may be potentially useful to enable their research and move it forward. A big advance in this field of repositories was the paper by Francis Collins and colleagues on the IKMC published in the journal Cell in 2006. A significant part, postproduction, of an embryonic stem cell library comprehensively covering every protein-encoding gene across the mouse genome is a repository network to be able to deliver that resource to the rest of the world. So KOMP, EUCOMM, NorCOMM, and the Texas Institute of Genomic Medicine have been established as an IKMC repository network.

NorCOMM has been funded by Genome Canada to establish a Canadian academic-industry lab network. The CMMR became the repository and distribution center for the NorCOMM resource. The project is working toward 2,000 conditional-ready targeted genes and 10,000 nonconditional trapped genes (genes mutated in embryonic stem [ES] cells using an alternative technology called gene trapping).

A component of the project is to create mouse lines from these ES cells. We are also performing functional analysis of candidate genes identified as important determinants of specific human disease. This is also funded by Genome Canada.

We are using two approaches to mouse mutagenesis or to ES cell mutagenesis. Gene trapping is a random process, inserting a vector randomly across the genome. The vector is tagged and so may be found as would the gene in which it was inserted. Another approach is gene targeting, a more focused approach where the insertion replacement relies on homologous recombination.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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The element is targeted to maximize the mutagenic potential in the specific gene that we are interested in.

The project is focusing on 866 genes on a prioritized list. The first 100 gene targets will be used for some quality control and to address some of the germline transmission issues associated with these ES cells, and 50 of these lines from our prioritized list will be those of specific Canadian interest—related to disease areas in which our research community is actively engaged.

NorCOMM has a website (www.norcomm.org) that describes our partners, production labs, repository, and target construction groups. It provides the opportunity for us to engage the Canadian as well as the international research community. Anyone has the opportunity to use our gene submission process.

Our gene prioritization process addresses high-impact Canadian health research community genes; those that are not targeted by EUCOMM or KOMP; those whose gene structure is amenable to our particular allele and our approach. We focus on the 2,000 genes that are available to us and are not being done by another consortium.

The genes available in the pipeline are being posted at the Wellcome Trust Sanger Institute (www.sanger.ac.uk/htgt) by KOMP, EUCOMM, and NorCOMM.

One can see from the pipeline summary how many genes are involved in each group, whether the design has been requested or, more specifically, if the vector construction is actually in progress. ES cells in the pipeline are also included. This is an evolution from the CMMR in that where we were doing tissue, germ cell, and embryo archives, we are now also responsible for the embryonic stem cells for the NorCOMM project.

Our main contribution in the international arena is the trapping technology. Each of the resources or production centers has been or is currently engaged in trapping some component of their unique alleles across the genome. We would like to trap 10,000 genes and currently hold about 90,000 cell lines. The target is to trap about 38,000 genes across the different centers with a significant number of cell lines. These repositories are large-scale infrastructures that will be able to support those production objectives.

For targeting the genome, we are focused on 2,000 genes at NorCOMM. The target for all the repositories is to hold ES cells representing over 18,000 genes, and working together we eliminate redundancies.

We have tried to increase accessibility by creating as many launching points into the NorCOMM resource as possible, through the international gene trap consortium or through all the Web portals. You get to the CMMR (hosted by the TCP website; www.phenogenomics.ca) and essentially move through a very simple process. Rather than a materials transfer agreement (MTA), we have instituted a conditions-of-use agreement for not-for-profit users, which is simply a checkbox stating that the user agrees to our online conditions of use. With for-profits, we still deal with MTAs. Once the user has agreed to the conditions, s/he makes a payment, and we can confirm and ship the clone.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

Requests for material are international, with the United States being a significant requester. In 2007 shipments went to 11 countries in Europe, Asia, and Australia in addition to North America.

There are challenges ahead for our repository that are shared by other repositories. For example, when ES cell resources need to be developed into mice, there is likely to be a parallel centralization of the production of these genetically engineered mouse models, or GEMMs.

The basis for the IKMC is to develop all the 22,000 protein-encoding genes in the mouse genome, in BL/6 ES cell lines. Because BL/6 ES cell lines present challenges with germline transmission, success in development, and culture conditions it is quite possible that not every lab or transgenic facility will achieve the same efficiencies with BL/6 ES cells that they have had with the more robust ES cells of the past. So if investigators come to rely on centralized facilities to create these mice, this would create issues in transporting live mice.

Sperm cryopreservation is also an option and there has been great progress in sperm efficiencies after cryopreservation. However, the preference for BL/6 background continues to be challenging because of the problems in sperm thawing and reanimation processes. While the Jackson Laboratory in particular has made great progress with this, the efficiency has increased only from 3% to 40%. This is not efficient enough for the needs of the international community, since even 40% efficiency is achievable only by experts and is likely to be lower in the hands of those more unfamiliar with the techniques.

I think sustainability and accessibility of these international knockout mouse resources (IKMRs) are challenges. For example, NorCOMM is funded in the same four-year lifecycle as the production. When the funding cycle ceases for the production, the repository is left with the resource, making its sustainability an issue. In addition, the resources will need to change as the science changes. The IKMC must also provide a quality, standardized, and secure resource; it must be innovative with its technology, procedures, and offerings; and it must establish partnerships, platforms, and processes based on sharing.

However, along with the challenges there are opportunities. One is comprehensive biobanking that goes beyond embryos and sperm, and moving to serum for blood-based biomarker or proteomic research. In addition, there are a number of techniques and technologies to provide the user with other ways to access these banks. Laser capture microdissection is one in which we can load up tissue sections and then purify and amplify RNA or DNA from specific homogeneous cell sets. For example, if the requester is interested in respiratory epithelium and wants to run some gene expression experiments on that, we can send the cells of interest or the purified RNA from the cells. Whole-tissue slide scanning is another important technology that can be applied to these repositories of ES cells or to mice. We use whole-tissue high-resolution slide scanning in the pathology setting and make the slides available to users around the world. We can interact with investigators while looking at the same slide and discuss the pathology phenotype that we are describing. The same technology can be applied for an investigator accessing the repository, i.e., looking at a model

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

that’s sitting in the archive to ask questions about the pathology to determine, for example, whether a mouse really needs to be transported.

Another development in comprehensive biobanking is the use of induced pluripotent stem (iPS) cells. The international collaboration and distribution of these resources will result in building better models and in reducing attrition, which relates to the number of mice moving around the world. We can induce skin fibroblasts with four genetic factors to become pluripotent stem cells, develop an allelic series of mutations across a gene of interest, and provide the tools to characterize them early, to assist investigators in making the decision about whether they really need this mouse model shipped around the world or not.

Also, for secondary modifications, again the models that we are characterizing, that we are depositing in our repository, have typically come through a phenotyping pipeline. They have information. They have engaged investigators somewhere. But maybe it is not exactly the mutation in their gene of interest that they need. I think the older approach would be to go back, retarget, pick your clones, create a mouse, expand—all the breeding that is required for that—germline transmission. We have the potential with iPS cells to have homozygous cells recovered from the skin fibroblasts, induced to iPS cells, make a secondary modification genetically, re-create a mouse, save all that additional effort, and ultimately save a lot of transport and distribution of mice.

To summarize the progression, we were taking tissue, sperm, ovaries, and embryos, and developing a relatively simple scheme for archive and distribution. Now ES cells have been added and we are working on iPS cells, banking serum and tissue, and creating accessibility tools and layers like laser capture microdissection to provide a molecular resource, or virtual slide consultation to provide a pathology resource and be able to make all these resources as exportable as possible, in appropriate formats.

I need to recognize my collaborators and colleagues: Janet Rossant, Geoff Hicks, who lead the NorCOMM project; and my very hard-working group in the lab at the CMMR. We are hosted at the Toronto Centre for Phenogenomics. Of course, we are working very much and embedded and learning from our international collaborators in the IKMC repository network.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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EUCOMM, the European Conditional Mouse Mutagenesis Program

Martin Hrabé de Angelis

My presentation will focus on the European Conditional Mouse Mutagenesis Program or EUCOMM, the European part of this international endeavor to knock out every single gene, and will go a little further to describe our program on mouse phenotyping and on the outcome and distribution.

The challenges for the future are very clear:

• Identification of all the functional elements (that term is used rather than genes because the definition of gene is very complicated)

• Generation of a mutation for every gene in the mammalian (mouse) genome, which only provides one aspect of the function of a gene, because an allelic series may be needed for full understanding

• Phenotyping of these mice, which is the most important and the most challenging task

• Linkage of the models to human diseases

• Archiving and dissemination

EUCOMM produces vectors, conditionally mutated mouse ES cells, and mutant mice as a public resource, available to everyone. The goal is to have 13,000 mutations in the ES cells, which comprise a mixture of knockout and homologous recombination in ES cells; 320 of them are being made into live mice as test cases, [and] those 320 will eventually go into a phenotyping pipeline, which we set up in Europe. There are also currently available 2,500 gene-trap clones and over 600 clones mutated by gene targeting.

The consortium is headed and coordinated by Alan Bradley and Wolfgang Wurst; Wolfgang is at our center and Alan is at the Wellcome Trust Sanger Institute. The consortium is spread around Europe, and each center has different tasks. While it is not expedient to fully describe all the centers here, the information is available at a common website (www.eucomm.org), which is very valuable and convenient. It is possible to order clones from the website by clicking

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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the database to find the gene of interest and then contacting the person in our center, who will send you the ES cell lines. Information for prioritization of genes may be obtained from eucomm@sanger.ac.uk.

A limiting factor at the moment is annotation of the genome. Making the vectors proceeds automatically; however, if the annotation is not done properly then it is very difficult to design the vectors. Distribution of the cell lines is done through our center and for the mouse lines it is through EMMA, the European Mouse Mutant Archive.

Intellectual property for the gene-trap lines is handled by the tech transfer unit at our facility and at the Wellcome Trust Technology Center for the targeted lines.

An example of how we are using the different areas of mutagenesis, phenotyping, and archiving is with the disease osteogenesis imperfecta, or brittle bone disease, which is a group of genetic disorders. We created a mouse from our mutagenesis screen carrying a point mutation for collagen-1, alpha-1 gene, which is similar to the human situation. We treated these animals with bisphosphonates that target the osteoclast; this is also similar to human treatment. The result is that we were able to partially cure the bone phenotype. However, the lethality stayed absolutely the same, which was quite surprising. At this point, the idea of the mouse clinic came into play.

The German Mouse Clinic offers systemic phenotypic analysis of mouse mutants on the basis of scientific collaboration and primary screening of more than 320 parameters—I call this a hypothesis-generation machine. It consists of a consortium of people who are specialists in their areas—they all have a satellite lab in our mouse clinic—from urology to clinical chemistry, dysmorphology. There are 14 different indication areas. At the end of the day, once we phenotype the mice, everybody comes together again to discuss it and generate hypotheses.

With regard to the osteogenesis imperfecta mouse, we found a lung phenotype and a heart phenotype, based on a metabolic pathway. Clinicians felt that these phenotypes were secondary, because of the affected bones; however, from our work we know they are primary. This is an example of how early systemic phenotyping in the mouse clinic can provide critical information. In this case the story must be rewritten to reflect the fact that it is a systemic disease, affecting not only the bones but also the heart and lungs, which are relevant to the early death of these animals.

A roadmap for a European strategy for research infrastructure was designed over the last five to six years in Europe. One of the projects I am coordinating is the Infrafrontier program. Infrafrontier is in a pilot phase of building up the European infrastructure for phenotyping and archiving. We plan to create more mouse clinics and scale up the activities in EMMA. A second mouse clinic has been created that works closely with industry in which drugs can be tested in a systemic way. Steve Brown then created one at MRC Harwell, UK, and now there are clinics at the Sanger Institute, in Barcelona, and in Monterotondo. There is one in Toronto as well, with Colin McKerlie. However, despite this

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

growth, the repositories and phenotyping services are not yet adequate to handle the thousands of knockouts available as well as thousands of alleles from other mutagenesis programs. Thus, it is our hope to raise money for the Infrafrontier project, to bring the right people together, be ready in three years to meet this challenge.

FIMRe, the Federation of International Mouse Resources, is one of the examples of archives on a global level. The problem was exchange of material and exchange of mice. We weren’t able to agree on a common strategy so we started these bilateral science contracts now between our center, the Medical Research Council (UK), Mammalian Genetics Unit (UK), the National Center for Scientific Research (CNRS) in France, as well as with the Jackson Laboratory and UC Davis in the United States, with RIKEN in Japan, and others.

The Infrafrontier consortium, which also takes part in FIMRe, consists of several countries. With the next amendment of the contract, Canada will join the consortium, as will the Academy of Sciences in the Czech Republic. The consortium consists of scientific labs and funding agencies that develop a plan for future endeavors. The goal is to have a very clear strategic plan by 2011, including a business plan for how to run the resource and a memorandum of understanding between all the different countries. The consortium is open to additional partners.

In summary, I have presented the European strategy for mouse mutagenesis, phenotyping, and archiving. The European knockout project, EUCOMM, focuses on mutagenesis and is only one part of the European roadmap. There are also other technologies, such as RNAi, that play a very strong role. There is also EUMODIC, the European Mouse Disease Clinic, a consortium headed by Steve Brown, where we have a pilot project combining mouse clinics. Another component is EMMA, where we have currently some 1,500 mouse lines, not counting the ES cell lines. In the next three years there will probably be 4,000 to 5,000 lines. Finally, Infrafrontier is one plan to come up with the proper infrastructure and funding for these different areas.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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The RIKEN BioResource Center

Yuichi Obata

The RIKEN BioResource Center (BRC) was established in 2001, making it relatively new in the field. It was established to produce an infrastructure for advancement of life sciences. The center focuses on five major activities:

• collection, preservation, quality control, and distribution of bioresources

• key technology development necessary for production, preservation, and distribution of bioresources

• bioresource frontier program, including a mouse clinic

• training and education

• international collaboration

We have focused on five major resources that include not only the mouse but also the experimental plant Arabidopsis as well as cell lines and genes from mammals and microbes and the microbes themselves. Finally, we focus on information itself as an important resource now and in the future.

RIKEN BRC serves as a station for dissemination of research products or bioresources produced by Japanese and RIKEN scientists to the international scientific community. All materials come in with material transfer agreements to protect the intellectual property rights of the developer and to ensure their proper use by the recipient.

Coinciding with the RIKEN BioResource Center, the Japanese Ministry of Education, Culture, Sports, and Science and Technology inaugurated the National Bioresource Project in 2002. The aim of this project is to distribute biological resources of the highest quality by 2010. The first term ended in 2006, but it will continue through a second term to 2010. Twenty-eight bioresources were selected for the project: 10 mouse and 9 plant bioresources, and 9 microbes and cell lines and others. RIKEN is responsible for five of these. These projects are run by a government committee and evaluated by an external review committee.

With respect to mouse strains, we now hold over 3,200, a quarter of which are traditional inbred or mutant strains, a quarter are transgenic mice, and a

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

quarter are knockout. About 20% are ENU (ethylnitrosourea) mutants, developed by RIKEN and the Korea Institute of Toxicology, and 3.5% are wild-derived mouse strains, which are unique to our bioresource center. Because of the genetic diversity of the commonly used laboratory mouse these mice are very useful for dissecting gene functions. They are also very popular with overseas users.

In addition to mouse resources, the RIKEN BRC distributes embryonic stem (ES) cell lines from C57BL/6 and in some of these the germline transmission is confirmed. RIKEN also has ES cells of inbred strains from nuclear transfer, mouse induced pluripotent stem (iPS) cells (as well as human iPS cells, developed by Dr. Yamanaka). In addition, 369 ES gene-trap clones and the Mus musculus molossinus (MSM) wild-type-derived mouse BAC (bacterial artificial chromosome) library are distributed from the gene bank at our center.

From 2004 to 2008, nearly 8,000 shipments were made, with 80% for domestic use and about 20% for international use. We distributed to over 20 countries; the most frequent users are the United States overall and Korea in Asia, followed by Germany and Belgium. Minimizing the shipping time for embryos and sperm is very important since they must be kept frozen. International shipping times range from 30 hours (China) to 81 hours (Italy); the longest shipping time to the United States is over 60 hours to Oregon. All mice are transported safely, but the cost of shipping the mice is unacceptable. Since international shipment of mouse strains is expected to increase drastically in the next few years, more economical transportation is needed for the global scientific community.

As a member of the Federation of International Mouse Resources (FIMRe), RIKEN can help to rederive a mouse strain sent from somewhere else in the world. For example, we help Japanese scientists with material from the Mutant Mouse Regional Resource Centers (MMRRC) or Jackson Lab.

For the RIKEN repository, the quality control of mouse strains is most important from deposition to distribution. All mouse strains are tested for microbial infection status and according to their status are housed in positive- or negative-pressure rooms for breeding. All mouse strains received are cleaned up by caesarean section or IVF. After confinement, if they are negative for all microbes, they go to a barrier facility for further production. Mice are tested for eight most hazardous microbes and viruses when they arrive and also bimonthly. Seven additional microbes and three parasites are tested for bimonthly. Three microbes that cause opportunistic infections are also tested bimonthly. Eight other viruses are tested at the request of our users. It has been very difficult to agree on a list of microbes to test since the infectious status is different from country to country and continent to continent. However, if cross-border shipping is going to increase, a list of minimum testing should be agreed upon by international mouse repositories.

Genetic quality control is also important. Our facility does allele-specific PCR and the genetic background is monitored by microsatellite markers. It is

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

important to have a uniform background, since the background of the mouse strains influences the phenotypes.

Since we have so many mouse strains to be developed, cryopreservation is very important. We now have 2,000 strains frozen as well as a backup facility 700 kilometers from the main campus, since Japan is an earthquake-prone country. Although shipping frozen material in a dry shipper is cheaper than shipping live mice, it is still expensive. We conducted a test with the MRC in the United Kingdom where we froze mouse testes at −80oC and shipped them to RIKEN in dry ice. We then rederived the mice by microinsemination. This process can cut the cost of the shipping but the facilities must be familiar with the technique of microinsemination.

Another function of our facility is providing training courses. The courses we currently offer are

• Experimental Animals: Cryopreservation of Mouse Embryos and Sperm

• Experimental Plants: Culturing Method for Plant Cell Lines

• Genes: Recombinant Adenoviral Vector

• Microbes: Culturing and Preservation Method for Anaerobic Microbes

• Terminal RFLP1 Method for Analysis of Intestinal Bacteria

These courses are offered to universities, nonprofit institutions, and for-profit institutions. We also have international trainees from other places in Asia.

Finally, I would like to talk about our collaborations with Asian institutions. We have bilateral memoranda of understanding (MOUs) with institutes in China, Korea, and Taiwan. Based on these MOUs, the scientists come into our center and our fellows go to their country to teach the techniques.

Also, three years ago the Asian Mouse Mutagenesis Resources Association (AMMRA) was created to promote the mouse mutagenesis project and facilitate access to mouse strains in Asia. Its goal is the use of mouse models for understanding the genome function and improvement of human health. The first meeting was held in 2006 in Shanghai, the second in 2007 in Nanjing, and the next will be in Korea.

_______________________

1RFLP, restriction fragment length polymorphism.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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Repository Issues—Lessons Learned

James Womack

This presentation will be in two parts. First, I will convey information I have learned from workshops similar to this, as well as some reports and recommendations, and evaluate how well we have been able to implement the past recommendations. Second, I will describe a positive lesson learned in my laboratory, which shows the value of repositories specifically. This was not with transgenic or genetically modified animals but a very valuable rat strain that was produced in the old-fashioned way.

The need for genetic repositories has existed for 100 years. When Little, Castle, Wright, and others started making inbred strains at the beginning of the last century, it was obvious that the years of breeding and the amount of money that was put into making an inbred strain of mouse certainly could not be wasted by letting that strain become extinct. Consequently, places like the Jackson Laboratory and later commercial institutions, such as Charles River, Taconic, and others, have served as repositories for these valuable strains.

The need for live animal repositories has now been replaced by cryopreservation technologies developed over the last 50 years. In 1990, the ILAR Committee on Preservation of Laboratory Animals was convened to discuss what could be done with repositories. Basically, the discussion addressed what could be done best with live animal preservation versus cryopreservation? A very nice set of recommendations resulted from the deliberations and appeared in ILAR News No. 32.

Although we had begun making transgenic mice, this was well before gene knockout technology was developed. At that time, there was no idea what a germplasm repository might look like today.

With the advent of genetic technologies, the NIH, through the NCRR and Child Health and Human Development, convened another workshop last year. The goal was to reexamine repositories for germplasm in light of what was on the horizon from genetic modification and all of its implications. The recommendations from that workshop were:

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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1. Encourage the development of high-throughput and scalable technologies for germplasm collection, evaluation, processing, and cryopreservation;

2. Establish multidisciplinary teams to develop new approaches to the collection, cryopreservation, and distribution of germplasm for high-priority translational species;

3. Support research on the biosecurity of cryopreserved animal germplasm, and the detection and elimination of laboratory animal pathogens that might compromise research findings;

4. Support research to address long-standing bottlenecks to cryopreservation of animal germplasm, such as cold shock, chilling injury, protocol optimization, male-to-male variation; and

5. Support novel “high-risk/high-return” preservation technologies that are not dependent on freezing or cryopreservation and break new ground.

In looking at the recommendations one notices that informatics and databases are never mentioned, even in 2007, when it was obvious that the numbers of unique germplasm resources would eventually number in the tens of thousands. We have already achieved these numbers with mice, but there is also a zebrafish mutagenesis project and the technology is beginning to be developed in rats. There will be tens of thousands of unique germplasms and yet not much attention has been given to the development of informatics and databases.

In my view as a user, this is a bottleneck. While the conclusions from the workshop were to make the biological materials readily available to biomedical investigators at low cost, most of us have no idea what is available. As we have learned from others at this conference, the major repositories for genetically modified mice—the targeted mutagenesis, the knockouts—are developing databases. But when you access them, if you know the name of the gene, you can find your knockout if it exists. However, if you are interested in a phenotype, as many of us are, unless these mutants are written up in a scientific publication that we can access through PubMed or some other way, they are essentially lost to the biomedical science community. The goal of most investigators is to learn which genes underlie a specific phenotype, thus they cannot search for a gene.

So as a user, my word of admonition would be that we look seriously at the future needs in informatics and databases to support these tens of thousands of mutant mice, rats, zebrafish, and any other species that will be included.

Two ILAR resources that are simply catalogues should be highlighted here. The first is a catalogue of available databases and search engines that each of the repositories has put out. This is a very useful place to begin searching. Second, ILAR has an animal model search engine, which begins to address some of the phenotypic information. However, the point remains that our ability to make useful laboratory animal resources is outstripping our database development and capacity at this time. Those of us who use resources may very well be located close to a resource that we need and never know it.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

The second part of my presentation consists of a quick real-life story—a positive one. It does not involve genetically modified mice but a unique germplasm resource. It is a nice story that is not quite finished.

Rift Valley fever is an infectious disease caused by a bunyavirus. As the name indicates, it was identified in the Rift Valley of Africa and the virus is mosquito borne. In the cycles of flooding that very often occur in central Africa and into the sub-Saharan region, there are extreme epidemics of Rift Valley fever. Tens of thousands of livestock are killed as well as some humans, although the human death rate is usually low. But the livestock industry is devastated.

C.J. Peters, who was at Fort Detrick in Frederick, Maryland, in the 1980s, isolated some of the major strains of Rift Valley fever virus and did some laboratory animal experiments. When he did a strain survey of rats, he found that the Lewis rat strain was resistant to the Rift Valley fever. All other strains that he infected died within several days.

In doing some simple genetics (i.e., typical backcrosses), the resistance segregated as a simple Mendelian trait. Resistance was dominant to the susceptibility in all of the strains. He then made a congenic strain—he backcrossed Lewis onto a Wistar-Furth background, each generation selecting after challenge, over a period of about three years, in which the resistant gene from Lewis had been placed on the Wistar-Furth background.

For a variety of reasons, funding was stopped. The NIH was not particularly interested in a livestock disease in Africa. C.J. put his congenic strain down as a frozen embryo resource with the animal germplasm resource bank at the NIH. It later was moved to the Rat Research and Resource Center at the University of Missouri where it sat for 15 years.

After 9/11/01 many things in this country and around the world changed, including some of our research interests. Suddenly we became concerned about biological terrorism and agricultural bioterrorism. Rift Valley fever was targeted as one of the diseases that we should learn more about because, if introduced accidentally or intentionally into this country, it would have a devastating effect.

Fortunately, C.J. had published a paper—through PubMed we were able to find that he had made this congenic strain of rat resistant to Rift Valley fever—and he is still doing research and was available. It was through personal communication that I learned that he had had the congenic strain frozen.

When I contacted the NIH and they directed me to the University of Missouri, I was told that, yes, the embryos had been there. They had been frozen for 15 years, and they could be brought out.

Genomic resources had changed tremendously in 15 years: now we had a gene map of the rat, DNA-level markers, and microsatellites that enabled us to do things that C.J. and others could not do back in the 1980s.

While the embryos were being rederived, we took a piece of tissue from these congenic strains and very quickly did a genome scan with highly polymorphic microsatellite markers. We tested across the genome with markers that had a high probability of distinguishing Wistar-Furth alleles from the Lewis alleles.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

Then we found that some of these were monomorphic as far as Wistar-Furth and Lewis were concerned: they carried the same allele.

When we looked at the congenic strain, even before the embryos had been rederived, we found Lewis markers on chromosomes 9 and 3. So in making the congenic strain, C.J. and his colleagues had incorporated the Lewis genome into the bottom of chromosome 3 and up near the top of chromosome 9. The question, of course, at this point is, Since it behaves as a Mendelian single gene trait, which of these two sites is actually the gene and which may be the hitchhiking material? We did a quick cross and determined that, in fact, it was the bottom of chromosome 3.

To date we have narrowed it down to a little bit more than a megabase. Almost all the genes in this region are transcription factors. We are now in the process of finding which of these is responsible for conveying the total resistance to the virus to the Lewis strain.

In summary, this is a model that was developed as far as the technology would allow in 1990. The investigators had the foresight to cryopreserve something that they no longer had funding to work with but felt was important. The technology and the repository for cryopreservation and maintenance and rederivation were all there and used very efficiently and very effectively.

Meanwhile, a tremendous battery of new genomic tools was developed. So when we rederived this model, we were able to narrow down to a megabase region and will very soon have the gene.

To go back to my original point, we found this model by good fortune. The phenotype was available in the literature and we found it by PubMed. But until we start getting phenotypic data into the databases that accompany many of these great repositories that we are seeing developing around the world we will not be able to use these resources to their full extent.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

Transportation and the “Mouse Passport”

William White

In this presentation I will discuss the “mouse passport” and key issues in the transportation of rodents, and propose some recommendations to remedy looming problems. The mouse passport is a product of the UK National Center for Replacement, Refinement, and Reduction of Animals in Research (NC3Rs; www.nc3rs.org.uk). It is not actually a legal document but rather a “detailed packing list, with assembly instructions and an operating manual.” The goal is to have a lot of information about the animals in one place. Some of the information in the mouse passport is nomenclature/lineage, background strain, number of backcrosses, whether it is inbred or outbred, type of mutant (knockout, chemical mutant, etc.), genotype, phenotype, immune status, animal husbandry details, breeding information, and special considerations.

It is important to provide enough details about genetically modified animals to establish a new colony. Often not enough information is included in publications describing the model. In my opinion, the current document needs further expansion to ensure that all the necessary information is captured; there needs to be a “fill in the blanks” approach to minimize subjective evaluations.

Moving to the subject of transportation, there are essentially two alternatives: moving live animals or some type of cryopreserved material. Embryos, sperm, and the like may be shipped, but the facilities at the receiving institution must be able to recover the live animal. This is an important consideration as is the health status of the animals in the receiving facilities. This process makes good sense if complex long-distance shipment is required. Rodent germplasm is transported in liquid nitrogen dry shippers, which are approved for air transport. Shipping frozen material minimizes health risk at the receiving institution. However, there are some drawbacks, particularly in terms of time. When germplasm is shipped, there is a time delay until founder animals are generated, typically about five weeks. There are certainly variable recovery rates, which means more material must be shipped and more implants need to be done. Shipping of frozen resources also assumes that the institution can recover and maintain the animals at a desired health status. And if the animal is reconstituted at a repository, live animals may still need to be transported somewhere.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

The key to shipping live animals is knowledge about the system and thoughtful planning. Whether the animals go by air or by truck, across a few states or, in Europe, from one country to the next, it is necessary to anticipate what might go wrong. It is critically important to understand the transportation system. Animals are being put into air commerce that moves a lot of other material, and animals tend to be at the bottom of the list in terms of volume and economic value. Therefore, it is up to the shipper to understand how things move from one point to the next and what the options are if shipments are to be successful.

Animals are transported in commerce every day, particularly laboratory animals between institutions. But the total number of all animals shipped represents a tiny fraction of all goods that are moved—well under 1%. By and large, the journeys are successful. That, obviously, depends on your measure of success. If the shipment is delayed a day, but the animals are still in good condition, it may still be considered a failure because it didn’t come on time. The overall transportation failure rate (even if the failure was not directly due to transportation), based on statistics from breeders, is about 0.07% out of about 2 million containers. This is almost equally split between air and land transportation (about 0.035% each). Involved in the failure rate is any condition that might be cause for rejection upon receipt—even the wrong sex of animals in the box or only one animal with an abnormality out of a group of animals in the container.

Many factors can affect ground transportation, but the most frequent is temperature control related to the thermodynamics and ventilation in the cargo compartment of the aircraft or vehicle. These parameters are based on how containers are loaded and the type of containers used, coupled with the animal mass in the containers. These factors influence the ambient environment surrounding the container and the effective ambient environment in the container, which affects the animals.

HVAC (heating, ventilation, and air conditioning) systems are not capable of rigid temperature and humidity control for a wide range of ambient conditions. These systems can break down; some companies use redundant HVAC systems in case of that happening. However, if the system breaks down in the middle of Montana, chances are slim for finding a place to repair it. Available ground transportation carriers may be regional or long distance in the United States and in Europe. The problem is there are not many choices because it is not a big business.

Another factor to consider is that commercial carriers may transport other perishable and nonperishable cargo from multiple institutions along with the animals for all or some of the journey. While the use of a dedicated truck is possible, it is expensive ($1.50 to $3.00 a mile a few years ago, charged on a round-trip basis). A shipment of animal containers large enough to fill a tractor-trailer driven across the United States costs about $20,000. Those prices have increased between 15% and 25% due to fuel charges.

Only 40% of the commercial air fleet in the world is capable of carrying animals and not all compartments in an aircraft may have appropriate environ-

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
×

mental controls. In a 747, the first two compartments are incapable of carrying animals; only the three compartments in the back can carry them, but this varies with the aircraft. Another problem is that there are mixed loads. For example, if several containers of rodents are shipped in the same compartment with a cargo of flowers that needs a lower temperature, the carrier will try to select a temperature range that is acceptable to all of the temperature-critical cargo. It is not possible to dictate tight temperature ranges or the animals will not fly.

Another issue with air transportation is that there is always a ground component. If arrangements have not been made to retrieve the animals, they might languish in the customs warehouse for quite some time.

With regard to air transport, it is important to remember the following:

• It is the fastest way to transport, even when there is a ground component.

• Live animal shipments account for less than 0.1% of all air cargo, and lab animals are an even smaller fraction of that.

• It may be necessary to enclose as many as 39 separate documents for transport under certain conditions; generally, however, it is less than a third of that. Errors and missing documents can stop or delay the shipment. When the carriers cross borders, if everything isn’t there, the shipment will not move.

• There is limited liability in air cargo. If two mice have an estimated value of $10,000, the airline is only liable for $100 if something goes wrong. You need to have other insurance to cover any loss.

• Many factors can affect air transport of animals. Pilots or airlines can refuse to carry animals. Some things, like the mail, human remains, domestic farm animals, etc., can bump lab animal shipments. So even though all the arrangements are made, there is no guarantee that the animals will be transported as scheduled.

• The shipper is ultimately responsible for the microbiological status of the animals transported by air or land. It is the shipper’s responsibility to pack the animals in a microbiologically secure container to prevent contamination in shipment.

• Similarly, the shipper is responsible for escaped animals. Animals can chew out of containers, particularly those being reused. Escaped animals have grounded 747s and the shipper must pay the per-hour charge while the plane sits on the ground until the animals can be retrieved and any damage to the plane repaired.

• Anticipate weather delays, temperature embargoes, and canceled flights. If a huge snowstorm is coming to Chicago and the shipment is going to be routed through it, don’t start the journey.

Some things are important to keep in mind regarding any animal transportation:

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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• Once an animal leaves your institution, you have limited control over the environment and handling.

• The only way to minimize the risks in shipping is by journey planning and anticipation of potential problems. It is important to evaluate the risks and act accordingly.

• Animal transportation is highly regulated. It is important not to make any assumptions about what is needed, especially when shipping internationally. These rules change regularly. The International Air Transport Association (IATA) revises the Live Animals Regulations (LAR) yearly, and publishes the Air Cargo Tariff (TACT) book, which is updated every 6 months and contains the tariffs and shipping standards.

• Animals will experience some stress in transit but there are too many variables to precisely control it. Occasionally, animals will become sick or die either during or after transit, which may or may not be the result of errors in transit. Working with the transportation provider and collecting and analyzing the facts, and not making assumptions, can help in developing preventive action to lessen the chances of recurrence.

• When receiving animals, assume that the outside of the container is contaminated and take appropriate steps. The outside of the box should be disinfected.

• Separation of species during transit is not achievable. You are going to be in the same microbiological space. Rodents from multiple sources may be transported in the same van delivering animals to and from the airport.

When shipping genetically modified (GM) animals, some countries require special documentation and approval to enter or move within the country. Occasionally, a phenotype can make the animals less tolerant of transport conditions or the animal might have special requirements. It is important to remember that there will not be precise temperature or other controls en route. However, in most cases, these animals can be shipped as normal animals, as long as there is no overt disease or debilitating phenotype. GM animals are not considered dangerous goods by airlines or other groups. There may be specific regulations in countries as to classifying and handling them, but as far as transit goes they are not considered dangerous goods.

Authoritative references and shipping documents include the TACT book and LAR produced by IATA. IATA’s Live Animals and Perishables Board sets the standards for air transportation, which are followed by 260 airlines. Many governments and international bodies use the LAR as the primary transportation standard.

It is essential to comply with the receiving country’s requirements, which may be determined by calling the consulate or through an export agent or your consignee. It is best to have the receiving party coordinate documentation and the ground transportation.

To minimize problems:

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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• do not reuse shipping containers;

• plan for at least 24 hours in delay;

• ship at the beginning of the week and remember to consider holidays—many people do not work on Saturdays and Sundays, and holidays may differ depending on the country;

• develop a detailed journey plan;

• don’t transport during temperature or weather extremes;

• arrange for airport pickup by the consignee; and

• use the most current editions of resources such as the LAR.

There are ways to make live animal transport work more safely and efficiently in the future. It is better to ship germplasm if possible, but if it is necessary to ship live animals certain things are needed.

The scientific community needs to engage the air carriers through IATA on issues of air carriage of lab animals. This should be done by building a relationship with a sustainable commitment to a continuing dialogue. It is not enough only to complain when there is a problem. To assist IATA and to cultivate a relationship with the carriers, it is necessary to develop proactive materials to present to the heads of airlines that help reinforce the concept that laboratory animals are important to the biomedical research community and are a legal and essential cargo.

It is important to have a strategy and structure on which to base this interaction, perhaps under the umbrella of a scientific organization or a consortium of organizations. This should be international in scope, which may suggest a role for the OIE. You need the participation of multiple stakeholders, not just a couple in one country.

Training materials for all those involved in the shipping of live animals are advisable. It is our responsibility to provide access to correct practices and help carriers to better understand the needs of the animals they are shipping. To this end, IATA, in conjunction with ACLAM, has produced an interactive DVD aimed at shippers of rats and mice that will be released shortly. IATA also has formal training programs for air carriers. However, collaboration there could be helpful. A similar program for ground transportation carriers is needed.

Another resource that is needed is an electronic master system for preparing required documents for international and national shipment, somewhat akin to a tax preparation program. The user enters certain required information and the system selects all the required documents and fills them out. Such a system would avoid a lot of delays in shipping. However, it needs to be developed in a way that allows it to be continually and rapidly updated. It might start with rodents, but then expand to some of the other common laboratory animals. It would need to be maintained by a stable organization and underwritten by fees and/or grants. The same system could assist in journey planning and provide worksheets to guide shippers through the required steps and considerations before putting the animals into the system. A system like that has actually been

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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developed in Germany; unfortunately, the author of that computer system suddenly died and it is no longer available, but it should be pursued again.

Another helpful resource would be the implementation of an “e-freight” system for lab animal shipments. This would allow all documents required for a shipment of animals to be paperless and to be sent for preapproval to catch any errors that might halt or delay shipment or importation. It would also address the issue of losses in shipment. This sort of system is being worked out for other types of air cargo. IATA is very supportive, and if the scientific community were to do the same and worked with them in developing it, it would go a long way in reducing errors in shipment.

Another consideration is the development of government-supported, academic-based, and commercial nodes for streamlined movement of animals. This would require a lot of organization and would need a variety of alternatives. Some of this is already available on a commercial basis and by cooperation between repositories. Key issues here are funding and access. In addition, there must be allowances for protection of intellectual property and downstream liability for errors in the process.

Last, there is the cold chain process used for shipment of critical products and ingredients. This monitoring process involves looking at the temperature and other environmental conditions of materials as they move through the transport system. Much of the information about transportation failures, especially with ground transportation, is anecdotal. An effort to proactively track environmental conditions and to work with transporters could be very helpful. This may be done with devices like the TurboTag, which will do 700 interval recordings of temperature and can be disinfected and reused. It is read with an RFID (radio frequency identification) reader and the readings are downloaded into an electronic record. Each TurboTag costs about $20; the reader is about $75. We have started putting them throughout shipments to look at airflows and temperature mapping. They will help us to get a better understanding of where failures are and how we can prevent them.

Suggested Citation:"Coordination of International Rodent Resources." National Research Council. 2011. Animal Research in a Global Environment: Meeting the Challenges: Proceedings of the November 2008 International Workshop. Washington, DC: The National Academies Press. doi: 10.17226/13175.
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Animal research will play an essential role in efforts to meet increasing demands for global health care. Yet the animal research community faces the challenge of overcoming negative impressions that industry and academia engage in international collaborations in order to conduct work in parts of the world where animal welfare standards are less stringent. Thus, the importance of ensuring the international harmonization of the principles and standards of animal care and use cannot be overstated. A number of national and international groups are actively working toward this goal.

The Institute for Laboratory Animal Research (ILAR), a program unit of the US National Research Council, is committed to promoting both the welfare of animals used in research and the quality of the resulting science. In 2008, to follow up on the 2003 event, ILAR convened a workshop which brought together 200 participants from 17 countries. Their mission was to identify and promote better understanding of important challenges in the conduct of animal research across country boundaries. These challenges include: the sourcing of animals; the quality of veterinary care; competent staff; the provision of a suitable environment (including nutritious food and potable water) for animals; and ongoing oversight of the animal program; among others.

Animal Research in a Global Environment summarizes the proceedings of the 2008 workshop. The impact of this 2008 workshop has extended beyond the oral presentations conveyed in these proceedings. It has been a vital bridge for diverse colleagues and organizations around the world to advance initiatives designed to fill gaps in standards, professional qualifications, and coordination of animal use.

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