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Coordination of International Rodent Resources

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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 re- sources, 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 re- stricted 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 noth- ing 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 patent- ing 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 re- pository. A general consensus is that this position promotes good citizenship in the life sciences community. 187

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188 Animal Research in a Global Environment: Meeting the Challenges As a matter of policy at NIH and many academic institutions, the technol- ogy 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 re- quester 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 prop- erty perspective, this criterion is of most value in these resources so it is neces- sary 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 deriva- tives and unmodified progeny, zygotes, embryos, and cells, tissues, etc. There is also language that allows for crossbreeding. This language is pri- marily for nonacademics, so they know NIH allows it and if you plan to distrib- ute 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 prop- erty, 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 fa- cilitate the transfer. Sometimes these license agreements define how you can further transfer these mice—for instance, research purposes only and to aca- demic 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 institu- tion. 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

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Mice Traveling the World: Issues in Sharing and Transferring Mice 189 the intellectual property and make sure it is okay to transfer it to another indi- vidual. 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 agree- ment 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 incorpo- rated 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 particu- lar 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 be- come 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-

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190 Animal Research in a Global Environment: Meeting the Challenges 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 knock- out, 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 paper- work to ship mice.

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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 differ- ent mutations that mimic pathologic or disease conditions seen in humans. In the past, spontaneous genetic mutations in mice have contributed to un- derstanding the biology of human disease in significant ways, but today’s focus has shifted to induced, genetically engineered, or modified mouse strains. Spe- cifically, 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 cen- tury 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 devel- opment 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 wide- spread 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 191

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192 Animal Research in a Global Environment: Meeting the Challenges 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) knock- out 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 (Nor- COMM) 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. Addi- tionally, 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 per- centage 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 gen- erated products as a result of the testing (e.g., germline-transmitted mice, em- bryos, and sperm) are archived as well. The repository allows the customer to learn about KOMP and its opera- tions, 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

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Knockout Mouse Databases: The Knockout Mouse Project and Repository 193 (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 interna- tional 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 pro- duced by one project would be available to scientists on a different continent, thereby enabling scientists to simply order all mouse strains locally, thus avoid- ing 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.

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NorCOMM, the North American Conditional Mouse Mutagenesis Project Colin McKerlie The focus of this presentation is NorCOMM, the North American Condi- tional Mouse Mutagenesis Project, in particular where it came from and the con- tribution of my laboratory, which is the archive and distribution program. In 2003, some collaborators and I at Mount Sinai Hospital Samuel Lunenfeld Re- search 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 pheno- typing 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 ana- lyzing, 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- 194

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NorCOMM, the North American Conditional Mouse Mutagenesis Project 195 vantage in terms of protection from a disaster as well as some additional oppor- tunities. 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 me- chanical 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 en- terprise 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 reposi- tory network. NorCOMM has been funded by Genome Canada to establish a Canadian academic-industry lab network. The CMMR became the repository and distribu- tion 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 im- portant 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.

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204 Animal Research in a Global Environment: Meeting the Challenges 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 coun- try. 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 institu- tions. 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 Associa- tion (AMMRA) was created to promote the mouse mutagenesis project and fa- cilitate 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. 1 RFLP, restriction fragment length polymorphism.

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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 rec- ommendations, and evaluate how well we have been able to implement the past recommendations. Second, I will describe a positive lesson learned in my labo- ratory, 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 cryopre- servation 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 recom- mendations from that workshop were: 205

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206 Animal Research in a Global Environment: Meeting the Challenges 1. Encourage the development of high-throughput and scalable technolo- gies for germplasm collection, evaluation, processing, and cryopreservation; 2. Establish multidisciplinary teams to develop new approaches to the col- lection, cryopreservation, and distribution of germplasm for high-priority trans- lational species; 3. Support research on the biosecurity of cryopreserved animal germ- plasm, and the detection and elimination of laboratory animal pathogens that might compromise research findings; 4. Support research to address long-standing bottlenecks to cryopreserva- tion of animal germplasm, such as cold shock, chilling injury, protocol optimiza- tion, 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 data- bases are never mentioned, even in 2007, when it was obvious that the numbers of unique germplasm resources would eventually number in the tens of thou- sands. 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 data- bases. 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 devel- opment 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.

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Repository Issues—Lessons Learned 207 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 germ- plasm 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 fe- ver. 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 labo- ratory 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 suscep- tibility in all of the strains. He then made a congenic strain—he backcrossed Lewis onto a Wistar-Furth background, each generation selecting after chal- lenge, 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 particu- larly 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 Univer- sity 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 com- munication that I learned that he had had the congenic strain frozen. When I contacted the NIH and they directed me to the University of Mis- souri, 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 polymor- phic microsatellite markers. We tested across the genome with markers that had a high probability of distinguishing Wistar-Furth alleles from the Lewis alleles.

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208 Animal Research in a Global Environment: Meeting the Challenges 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 ques- tion, 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 resis- tance 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 some- thing that they no longer had funding to work with but felt was important. The technology and the repository for cryopreservation and maintenance and rederi- vation 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 re- gion 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.

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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 informa- tion 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 ani- mals 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 alterna- tives: 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 germ- plasm is shipped, there is a time delay until founder animals are generated, typi- cally 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 reposi- tory, live animals may still need to be transported somewhere. 209

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210 Animal Research in a Global Environment: Meeting the Challenges 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 mate- rial, 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 repre- sents 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 suc- cess. 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 transporta- tion), 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 con- tainers 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 condi- tions. 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 pos- sible, 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-

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Transportation and the “Mouse Passport” 211 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 tem- perature 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 com- ponent. 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 compo- nent.  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 ar- rangements 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 trans- portation:

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212 Animal Research in a Global Environment: Meeting the Challenges  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 disin- fected.  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 re- quire special documentation and approval to enter or move within the country. Occasionally, a phenotype can make the animals less tolerant of transport condi- tions 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 dan- gerous 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:

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Transportation and the “Mouse Passport” 213  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 effi- ciently in the future. It is better to ship germplasm if possible, but if it is neces- sary 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 rela- tionship 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 culti- vate a relationship with the carriers, it is necessary to develop proactive materi- als to present to the heads of airlines that help reinforce the concept that labora- tory 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 inter- action, 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 cou- ple 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 prepar- ing 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 ro- dents, 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 be- fore putting the animals into the system. A system like that has actually been

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214 Animal Research in a Global Environment: Meeting the Challenges developed in Germany; unfortunately, the author of that computer system sud- denly 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, aca- demic-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 be- tween repositories. Key issues here are funding and access. In addition, there must be allowances for protection of intellectual property and downstream li- ability 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 trans- port system. Much of the information about transportation failures, especially with ground transportation, is anecdotal. An effort to proactively track environ- mental 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 elec- tronic 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.