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Mouse Bank at CARD Kumamoto University, Japan
Naomi Nakagata

1,*1

Division of Reproductive Engineering, Center for Animal Resources & Development (CARD), Kumamoto University, 2-2-1
Honjo, Kumamoto 860-0811, Japan
Subject areas: Resources*Correspondence and requests formaterials should be addressed to N.N.
(nakagata@gpo.kumamoto-u.ac.jp).
Reviewer: Chi-Kuang Leo Wang,
National Laboratory Animal Center,Taiwan; Atsushi Yoshiki, RIKEN
BioResource Center, Japan
Editor: Gonsup Kim, Gyeongsang
National Univeristy, Republic of KoreaReceived December 16, 2010;
Accepted December 24, 2010;
Published December 30, 2010
Citation: Nakagata, N. Mouse Bank at
CARD Kumamoto University, Japan.
IBC 2010, 2:16, 1-4.
doi: 10.4051/ibc.2010.2.4.0016
Competing interest: All authors
declare no financial or personal
conflict that could inappropriately bias
their experiments or writing.

SYNOPSIS
Cryopreservation of mouse embryos and spermatozoa has become the foremost
technique for preserving large numbers of different strains of mice with induced mutations.
In 1998, our mouse bank was established in the Center for Animal Resources andDevelopment (CARD), Institute of Resource Development and Analysis, Kumamoto
University, Japan, based on the Preservation, supply and development of geneticallyengineered animals report published by the Ministry of Education, Culture, Sports, Scienceand Technology. We cryopreserve mouse embryos and sperm, supply these resources,
organize training courses to educate people and form part of a domestic and international
network of both mutagenesis and resource centers. We currently have over 1,500 mousestrains, 842,000 frozen embryos and 26,000 straws containing frozen sperm. Moreover, we
disclose information about 1,300 deposited strains. Furthermore, over 400 strains of frozen
embryos or mice produced from frozen embryos and sperm are being supplied to therequesters both domestically and internationally. Additionally we hold training courses on
the cryopreservation of mouse germplasm 2~3 times a year, both domestically andinternationally. In the course, we teach basic reproductive engineering techniques totrainees on a man-to-man basis. We have already held 28 training courses on thecryopreservation of mouse germplasm at our center and at other institutes.

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Keywords: resource, genetically engineered mouse, cryopreservation, embryo, sperm

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Introduction
The Center for Animal Resources and Development (CARD),
Kumamoto University was established in 1998 based on the“Preservation, supply and development of genetically engineeredmice” report published on July 10th, 1997 by the Subdivision of
Resource Study, Council for Science and Technology, Ministry of
Education, Culture, Sports, Science and Technology (available at
http://card.medic.kumamoto-u.ac.jp/card/english/index.html).
In 2000, a new facility for this purpose was completed and we
started to cryopreserve, supply and develop genetically engineered
mice. This paper attempts to introduce our mouse bank.
11) Mouse Bank1)-1 Mouse bank system
Figure 1 shows the outline of the mouse bank at our center.
When the researchers deposit their mouse strains, they send themice to our center. In our center, we carry out in vitro fertilization
using sperm and oocytes collected from the mice, then freeze theembryos produced and sperm in a liquid nitrogen tank. As a quality
control measure, a number of frozen embryos are thawed to check1) if the frozen embryos develop into live young, 2) whether thedeveloped mice are microbiologically clean, and 3) whether
transgenes are successfully transmitted to the young. We also thaw
one straw containing frozen sperm to check the sperm motility.
Thereafter, information regarding the deposited mice is put on ourwebsite (CARD R-BASE).
1)-2 CARD R-BASE
Figure 2 shows the website (CARD R-BASE: http://cardb.cc.
kumamoto-u.ac.jp/transgenic/index.jsp) concerning mouse strainsdeposited in our center. Mouse strains are classified as Inbred,
Spontaneous/Chemical induced mutant, Transgenic, Targetedmutant, Gene trap and Insertion mutant. If you click on individual
group of mouse strains, for example, „Transgenic‟, you can obtain alist of transgenic mouse strains. Moreover, if you click on eachstrain name, you can see more detailed information, such as strain,gene and reference.

Figure 2. CARD R-BASE website. Only part of the website is shown in thisfigure. Both Japanese and English versions are available.

1)-3 Supply of mouse strains (Figure 1)
First, requesters access CARD R-BASE. If they would like toobtain certain mouse strains, they order them from our center. If therequesters have adequate facilities to carry out reproductiveengineering techniques, we send frozen embryos. On the otherhand, if they do not have such facilities, we produce pups fromfrozen embryos or sperm, and the pups produced are sent to therequesters.
Using reproductive techniques, we manage the mouse bank very
efficiently (Figure 3). We currently have over 1,500 mouse strains(Table 1), 842,000 frozen embryos and 26,000 straws containing
frozen sperm. Moreover, we disclose information about 1,300
deposited strains (Figure 2). Furthermore, over 400 strains offrozen embryos or mice produced from frozen embryos and sperm
are being supplied to the requesters both domestically andinternationally (Table 1).

Figure 1. Outline of mouse bank at our center. Information on theapplication procedure and necessary documents are available from our website.
Mouse embryos or sperm obtained from mice sent from depositors arecryopreserved and stored in liquid nitrogen tanks. These frozen embryos, or live
mice obtained by transfer of thawed embryos into foster mothers, are sent torequesters.

Figure 3. The mouse bank system. The mouse bank system offers efficientand regular delivery of genetically engineered mice using reproductive engineering
techniques.

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Table 1. Cryopreservation and supply of genetically engineered
mice at CARD
No. of strains
cryopreserved

No. of strains
supplied

2000

144

4

2001

97102002

6734

2003

8933

2004

11658

2005

109392006

151

422007

169

35

2008

15958

2009

231

68

2010

221

61

Total

1,553

438

then released in 2007. This CD contains lots of pictures, photos andmovies to illustrate each topic item, and so is a useful tool forbeginners.
Contents
・Preparing and Assembling Pipettes for Embryo Handling・In Vitro Fertilization
・Simple Vitrification of Mouse Embryos
・Vitrification and Transplantation of Mouse Ovary・Cryopreservation of Mouse Spermatozoa
・In Vitro Fertilization using Cryopreserved Spermatozoa
・Partial Zona Dissection (PZD)
・Collecting Two-Cell-Stage Embryos
・Transportation of Mouse Oviducts Containing 2-Cell Embryos
at Low Temperature (0°C)
・Vasectomy for the Creation of Sterile Males
・Embryo Transfer into the Oviduct
・Production of Chimeric Mice by 8-Cell Aggregation
・Embryo Transfer into the Uterus
・Caesarean Section and Fostering2) Training Course on the Cryopreservation ofMouse Germplasm
We hold training courses on the cryopreservation of mouse
germplasm 2~3 times a year, both domestically and internationally.
In the course, we teach basic reproductive engineering techniques
to trainees on a man-to-man basis.
We have already held 28 training courses on thecryopreservation of mouse germplasm at our center and at otherinstitutes. Figure 4 shows the schedule for a training course thattook place at Bio-Evaluation Center, Korea Research Institute ofBioscience and Biotechnology (KRIBB), Chungbuk in April, 2008.
Among other things, we taught the trainees how to freeze spermand embryos, carry out in vitro fertilization, and transfer embryos.

3) CD Manual Regarding Reproductive Engineering
Techniques in Mice
The first CD edition of the manual for reproductive engineering
techniques in mice was released in 2005. The second edition wasUp to now, we have released a CD manual regarding
reproductive engineering techniques in mice in Japanese, English,
Chinese and Korean (http://www.funakoshi.co.jp/export/product/TRG/RETM-E.pdf). We plan to release revised versions containing
improved and new methods sequentially.

Conclusion and Prospects
Recently, we have developed a cryopreservation method formouse sperm to obtain a relatively high fertilization rate2,3. If wefreeze mouse sperm using this method, we can obtain over 1,000pups derived from frozen-thawed sperm collected from just one
male mouse via in vitro fertilization and embryo transfer techniques.
We also have demonstrated that the frozen-thawed 2-cell embryos
and the 2-cell embryos produced by frozen-thawed sperm andtransported from our center to many institutes at a refrigerated
temperature have the ability to develop well into live young4,5(Figure 5). The advantages of this method are that the receivers do
not have to master thawing techniques, and special containers (e.g.,dry shippers) are not required for the transport of embryos.
Therefore, I strongly believe that the transport of embryos at a cold

Figure 5. Live young derived from 2-cell embryos transported at arefrigerated temperature. Pups obtained from 2-cell embryos produced byFigure 4. Training course at Bio-Evaluation Center, Korea ResearchInstitute of Bioscience and Biotechnology (KRIBB). This figure shows
the course schedule and a ceremonial photograph.

frozen-thawed sperm transported at a refrigerated temperature from CARD,Kumamoto University to Asahikawa Medical College (a distance of over 2000 km)
are shown.

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temperature will become an established method for the exchange ofvarious strains of genetically engineered mice.
Since the establishment of our center, one of our goals has been
to set up a domestic and international network to promote biologicalsciences throughout the world. To accomplish this goal, we becamea founding member of the Federation of International Mouse
Resources (FIMRe: http://www.fimre.org/)6. The FIMRe is acollaborating group of Mouse Repository and Resource Centersworldwide whose collective goal is to archive and provide strains ofmice to the research community as cryopreserved embryos andgametes, ES cell lines, and live breeding stock. In addition, weorganized the Asian Mouse Mutagenesis and Resource Association(AMMRA: http://www.ammra.info/) in 2006. The AMMRA is acollaborative group of Mouse Mutagenesis and Resource Centers inAsia. Its mission is "To promote mouse mutagenesis projects and tofacilitate access to mouse resources in Asia". Its goals are “the useof mouse models for understanding the genome function andimprovement of human health".
Looking to the future, we would like to use new reproductiveengineering techniques in collaboration with the network describedabove in order to further develop our mouse bank and to provide
various genetically engineered mouse strains promptly to researchcommunities all around the world.

Acknowledgements

Haruguchi, Y Takeshita, Y Nakamuta, H Matsunaga, T Kondo and Y
Nakagawa, for their invaluable technical assistance.

References
1. Nakagata, N., and Yamamura, K. (2009). Current activities of
CARD as an international core center for mouse resources. Exp
Anim 58, 343-350.
2. Nakagata, N. (2011). Cryopreservation of mouse spermatozoa
and in vitro fertilization. Methods Mol Biol 693, 57-73.
3. Takeo, T., and Nakagata, N. (2010). Combination medium ofcryoprotective agents containing L-glutamine and methyl-{beta}cyclodextrin in a preincubation medium yields a high fertilization
rate for cryopreserved C57BL/6J mouse sperm. Lab Anim 44,
132-137.
4. Takeo, T., Kaneko, T., Haruguchi, Y., Fukumoto, K., Machida, H.,
Koga, M., Nakagawa, Y., Takeshita, Y., Matsuguma, T.,
Tsuchiyama, S., et al. (2009). Birth of mice from vitrified/warmed
2-cell embryos transported at a cold temperature. Cryobiology
58, 196-202.
5. Takeo, T., Kondo, T., Haruguchi, Y., Fukumoto, K., Nakagawa, Y.,
Takeshita, Y., Nakamuta, Y., Tsuchiyama, S., Shimizu, N.,
Hasegawa, T., et al. (2010). Short-term storage and transport atcold temperatures of 2-cell mouse embryos produced bycryopreserved sperm. J Am Assoc Lab Anim Sci 49, 415-419.
6. Davisson, M. (2006). FIMRe: Federation of International Mouse
Resources: global networking of resource centers. Mamm
Genome 17, 363-364.

I wish to thank Dr. T Takeo, S Tsuchiyama, K Fukumoto, Y

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