| Plans for the second 5 year term |

With the support of the CDB and NBR (National Bioresource Project), we are collaborating with researchers within the CDB and around Japan in developing mutant mice for research in the fields of developmental biology and regenerative science. As part of this collaboration, we have a responsibility for making mutant mice, while partner researchers for identifying genes of interest and analyzing mutant phenotype. We ask that ownership of the generated mutant mice remain with the CDB so that they can be freely distributed to researchers around the world upon request, while maintaining the condition that partner researchers can use the mice freely at any time for their own research, including collaborative work done with a third party. In principle, after two years have passed since the first publication using the specific mutant mice, we will then freely distribute the mice. Until that period has been reached, any mice generated are distributed to a third party only with the concurrence of our partner researchers. Please refer our homepage (http://www.cdb.riken.go.jp/arg/) for further details of the contract for use with our partner researchers.
We usually deliver germline chimera at weaning to our partner researchers within 8 months of receiving cDNA sequence information from them, or within four months of receiving the targeting vector. Mice born after DNA injection into zygotes are delivered to our partner researchers at weaning within three months of receiving transgene DNA from them.

In generating mutant mice by homologous recombination in ES cells, we will work by (i) generating targeting vectors with the sequence information provided by researchers, (ii) isolating homologous recombinants with targeting vectors made by researchers, or (iii) by producing chimera with homologous recombinants isolated by researchers. Option (iii) is only available to CDB researchers, and it is currently our operating principle to perform ES injection into embryos only when the cells are diploid after making karyotype examination in our hands. For option (ii), we will accept isolation only in cases where the vector is constructed under our supervision.
In our system one technical assistant constructs one targeting vector every two weeks. We now have two technical assistants and can make up to 40 vectors a year. We began the vector construction for collaborative mutant production on October 1, 2003 (Record 1). The targeting vector is constructed by isolating genomic sequences with long PCR where the gene is disrupted; it is constructed by BAC-based recombination where the gene is replaced with another gene or is conditionally mutated (Please refer to our homepage (http://www.cdb.riken.go.jp/arg/) for further details of vector construction). We currently have two technical assistants working on isolating homologous recombinant ES cells. One assistant can routinely isolate the ES recombinants at the rate of one vector per week, meaning we can isolate the recombinants at a rate of around 80-100 vectors a year (Record 2).
Zygotes are routinely frozen and these frozen zygotes are used as a source for host embryos for ES injection. One line of ES cells is routinely injected into 80 embryos at one time; four cell lines are injected each day, and injections are made on three days every week. It is therefore possible to inject 12 ES cell lines into about 1,000 embryos every week, with the embryos being transferred into about 50 foster mothers. Our capacity of injection is approximately 50 ES cell lines per month and 500 ES cell lines per year (Record 3). In our experience of the past three years, the injection has been made once in about half of the ES clones; germline chimeric mice are obtained on average in 5-10% of the embryos transferred to foster mothers. The injection has to be repeated, however, in around half of the ES clones to get germline chimeric mice. We have also produced chimeric mice with mutant ES cells isolated by CDB researchers. Generally, however, very few germline chimeric mice have been obtained with these ES cells. In January 2004 we introduced karyotype analysis for all ES clones, with only diploid clones being injected into host embryos to make germline chimeric mice

The production of transgenic mice by microinjection of cloned DNA into zygotes is a service provided for CDB members; this is done as collaborative projects for researchers outside CDB. We routinely inject one transgene into 250 zygotes which are fostered into 12 pseudopregnant females. We use frozen zygotes and administer the injections four days a week. We can therefore inject 4 constructs per week, 15-20 constructs per month and 150-200 constructs per year. We are currently able to carry out injection within two weeks of receiving a transgene DNA. We only accept, however, collaborative work requests for enhancer analysis from CDB members and researchers that belong to institutes with which the CDB has an official agreement on scientific exchange. The frequency of transgenesis is around 3% of zygotes injected in enhancer analysis (Record 4). For this analysis we instruct the careful preparation of the DNA solution to CDB members. In contrast to this, in the production of transgenic mouse with DNA solution from a variety of researchers, the efficiency is fairly low (Record 5). We also produce BAC transgenic mice (Record 6).

As an alternate, simple and/or more efficient method of cryopreservation of mouse strains, we have been examining the potential advantages of gamete freezing. Sperm is frozen by simply dipping them into liquid nitrogen. Sperm frozen in this way from several inbred strains (B6, BALB and CBA) are thawed by maintaining them in room temperature for about 5min and directly injecting them into unfertilized eggs of their respective strains. Fertilized eggs have been obtained at a rate of 1/2-1/3 of those obtained using fresh sperm; the rates are, however, strain- dependent, and is approximately 15%, even using fresh sperm, in the B6 strain. This dependency is most probably found in oocytes rather than in sperm, as the rate is fairly high at about 50% with both fresh and frozen B6 sperm when oocytes are BDF1. Current research is focusing on attempts to increase the rate of fertilization using B6 oocytes.
Ovaries are mildly minced and cryopreserved by vitrification. After thawing, they are transplanted into recipient females, and the females are mated naturally with males. We are currently investigating the mouse strain dependency of the success rate. Developing cloning technology for preservation of mouse strains is another issue. Earlobe and tail that are dissected upon mice identification and genotyping are frozen. The frozen tissues are thawed and cultured. Nuclei are isolated from the cultured cells and transplanted into unfertilized eggs, so that the mouse strain is reconstructed from frozen somatic tissues. The efficient isolation of nuclei of fibroblastic cells from these tissues is critical, and work is currently in progress to overcome this difficulty.

With mutant ES cells, mutant mice are obtained as F1 offspring of chimeric mice generated by the injection of cells into host embryos and transplanting the embryos into a foster mother. We have tested the possibility that nuclear transfer from mutant ES cells into enucleated eggs facilitates production of mutant mice. The success rate of nuclear transplantation in our process is approximately 3% of embryos transferred into the uterus of foster mothers with cumulus cells and with wild type ES cells. Nuclear transplantation with mutant ES cells was also performed at a similar success rate. The transplantation, however, was unsuccessful with ES clones that did not give rise to germ-line chimera (0/7). Moreover, only five among 21 ES clones that colonized into germline were successfully nuclear-transplanted when examined on 50 embryos that were transferred to foster mothers.

From fiscal year 2005, we have started to develop mouse strains by ourselves for wide use in mammalian genetics and embryology. We accept suggestions by researchers within the CDB and around Japan. The first mutants we are generating harbor a mutant loxP in Rosa locus (RosaloxP). Upon DNA injection into zygotes, transgenes are integrated randomly, with their expression dependent on the integrated sites. With the use of the RosaloxP zygotes as a host upon DNA injection, any transgenes will be integrated into the RosaloxP site efficiently by Cre-mediated recombination. Rosa locus is ubiquitously active, and the transgenes will be expressed ubiquitously. We are also developing mouse mutants that harbor a mutant loxP in a transcriptionaly neutral site to express transgenes spatio-tempo-specifically.
It is useful to be able to temporally trace the spatial relationship of different cell populations with embryogenesis. In collaboration with Dr. Hatta of Vertebrate Body Plan Group we have managed to generate mouse mutants that express Kaede ubiquitously. The color of Kaede changes from green to red by photo-conversion, thereby making it possible to trace the spatial relationship of different cell populations with embryogenesis.

This laboratory is also in charge of animal housing. In the CDB animal facility 32 rooms are available for animal housing, and four rooms for animal experiments. Each room is 25 meters square and contains 8 cage racks holding 72 cages. Water is supplied automatically, while cages are changed and cleaned, and food is supplied by our staff; 19 employees of JAC (Japan Animal Care) are in charge of this service. To minimize the chance of infectious diseases in housing animals, usually one housing room is used by members of only one project. At the end of March, 2004, two of the 32 rooms are shared by groups of small-scale users for mice and rats, respectively. Twenty eight rooms are used by each single project and two rooms are vacant. The number of rooms in which the number of mouse cages used is more than 90% of the maximum (576 cages) is three, twenty-five rooms are used at 50-80% capacity, and the number of rooms with less than 50% capacity is two. (Record7)
We are routinely freezing zygotes to stock mutant mouse strains by the vitrification method. We also routinely perform in vitro fertilization and transfer of fertilized eggs to foster mothers in order to expand mouse colonies quickly to meet the requests of CDB researchers. After receiving researchers' requests we are also able to routinely supply pregnant females, foster mothers and frozen embryos. Moreover, we are responsible for the exchange of mouse strains with institutes both inside and outside Japan.
(Record8-11)
For the welfare of experimental animals, we ask ourselves, as well as CDB researchers to ensure: (1) the healthy housing of animals, (2) minimization of animal pain and fear when used in experiments and (3) restriction of animal experiments to only those which are essential. In order to ensure this, a guideline for animal experiments has been prepared by the CDB Animal Experiment Committee, and we follow this guideline. The guideline is comparable to “Guide for the Care and Use of Laboratory Animals” by ILAR of the NIH. Researchers engaged in such experiments are well educated in them as set forth in the guideline.
Infectious diseases are one of the most critical issues, not only for the quality of animal experiments but also for animal welfare. Please refer to our homepage (http://www.cdb.riken.go.jp/arg/policy_en.html) for our policy and measures against infectious diseases. Propagation of infection is frequently accelerated by researchers, and we do not, therefore, allow researchers who are engaging in experiments with rodents in other facilities to enter the CDB animal facility. The exchange of animals with other facilities is another risk factor for contamination. With the exception of animals from breeders, we do not allow any live animals directly into the housing rooms even if they are certified by a veterinarian of the sender's institute; all animals are placed in the housing rooms only after cleaning by in vitro fertilization. We therefore ask senders to provide us with two to three males; moreover, the in vitro fertilization process benefits CDB researchers by the quick establishment of sufficient mouse colonies for experiments. When we send mice housed in the CDB to other facilities, they are accompanied by the health record of animals which have been housed in the same room. We consider it is the responsibility of the recipients, however, whether to agree to receive the mice from us and as to how they acclimatize the mice to their facility. We strongly recommend that they receive frozen embryos from us or introduce our mice into their facilities by in vitro fertilization; we are prepared to teach these techniques to the employees of any non-CDB facilities.