トップ > オッシー研究人生の軌跡と奇跡(English)

 The Miraculous History of My life as a Scientist
: A Great Journey to Medical and Biological Engineering of Chromosome  Born Out of Many Encounters

Mitsuo Oshimura
Head of Chromosome Engineering Research Center, Tottori University

‘Medical and Biological Engineering of Chromosome’ is a novel area of investigation that was originated in Tottori University (Japan) in which I had promoted research on chromosome science during the past quarter-century. The developmental process of this novel area is highly-overlapped with my life as a scientist. As a living witness to the birth and development of this novel area, I here trace my history as a chromosome scientist, which would lead the reader to understand of ‘Medical and Biological Engineering of Chromosome’. I would tell the history as accurate as possible, by tracing my memory.

The dawn
My encounter with ‘Chromosome’ stems from encounters of two key persons during my undergraduate days. One is Prof. Ken-Ichi Wakahama (Department of Literature and Science, Shimane University ), my supervisor, who was studying on chromosomes in flies. The other is Prof. Tsutomu Haga (Department of Science, Kyushu University), who was invited as a speaker in a special seminar by Prof. Wakahama at 1968 (my second year in university). The topic presented by Prof. Haga was morphological analysis of karyotype evolution in Drosophila and Trillium smallii. The scene that he was happily talking the topic with his own finding at that time is always on my mind. The presented topics were extremely sensational to me, since I understood that morphological analysis of chromosomes gives an important clue to know the process of evolution on a species of interest and that across the species. One of episodes of him that I heard later was that he sometimes gave a lecture over drinks. I am not sure whether that he was taking happily was due to drinks or not. One might say that this cannot or must not happen. Please be tolerant of it, since this episode happened in good old days.
 At 1969 (my third year in university), the student unrest broke out, which started from the battle demanding for a better deal of students in medical course occurred d at Yasuda Auditorium in University of Tokyo. This student movement had extended on a nationwide scale, and at last to Shimane University that I was studying. Political agitation in Shimane University, political activities in the city, and occupation of lecture room by ‘All-Campus Joint Struggle League’ lasted for half a year. As a corollary, the students had no class during this time. In my opinion, the most common purpose among the students was the opposition to authority, although the motivation differed among individuals as well as among universities. The attitude and behavior also differed among individuals. Some discusses how they should conduct the students and what the future of Japan should be. Some work hard to save money as a part-timer, or to play mah-jongg, and some went back to hometown.
Since I had my non-political point of view, I asked above-referenced Prof. Wakahama to give an opportunity to assist his research on chromosome research in Drosophila while I had no class. What I could assist at the most was to develop films in which he captured salivary chromosomes of Drosophila, to print them out, to prepare feed for Drosophila, and so on. However, similar to the way ‘children are prone to be influenced by their surroundings’, I got to be able to understand step by step what and how he promoted the research. Also, in the laboratory, I saw the Japanese Journal of Genetics (latter ‘Genes & Genetic Systems’) published by the Genetics Society of Japan that was in the possession of the laboratory. I sometimes paged through the journal and noticed the existence of Zoological Institute, Faculty of Science, Hokkaido University, whose researchers frequently reported chromosome studies on human and other animals in this journal. I got a feeling that this institute was a leading one on animal chromosome study.

Hokkaido University Era
When I was in the process of graduating from Shimane University, my future plan remained undetermined. Since I reckoned that I did not have unique talent and my elder brother and sister were school teachers, I had planned an ambiguous future that I would be a science teacher at the junior high school or high school or something (so called ‘a teacher for lack of anything better to do’). In fact, I had completed a curriculum for teaching certificate by then. For good or bad, Tottori prefecture, my home town imposed a freeze on recruitment of teacher at that time, and thus I did not fully prepare for the examination. In the meantime, I got an urge to pass into the world of animal chromosome study at Hokkaido University before I become a teacher. In this way, I became a disciple of Dr. Motomichi  Sasaki, Professor and Head of Zoological Institute, Faculty of Science, Hokkaido University. It was April, 1971, when I was 22 years old.
My first subject of research given by Prof. Sasaki was chromosome analyses of induced aborted fetuses. I started it with seniors’ supervising. I analyzed the chromosome by microscope observation for hours day after day. These research results had a substantial payoff in the form of international standard data to evaluate the frequency of chromosome abnormalities.
At that time, the time when I was allowed to study was only after noon, since my main work before noon was washing of equipments used by supervisors and seniors, a duty work of new faces that were transmitted year after year. After I acquired the skills of chromosome analysis, I was given second subject of research by Prof. Sasaki, which was study of venereal tumors of the dog. Stray dogs were seen everywhere, also in the campus of Hokkaido University. Motivation to this study of Prof. Sasaki was to know the reason why venereal tumors of stray dogs were observed rather specifically in the beginning of spring and autumn. I also got venereal tumors of stray dogs from Nagasaki by requesting a senior who had graduated from the same laboratory. We performed chromosome analyses and transplantation experiments that were conducted as a collaborative study with Faculty of Veterinary Medicine, Hokkaido University. We found that venereal tumors of the dog were propagated via the copulation. Moreover, by the comparison with the overseas cases, we clarified that these tumors in the world were originated from that of one dog. This study was reported at 1973 in Journal National Cancer Institute (JNCI), an international journal published from National Cancer Institute, and this was my first international publication.
This study required the analyses of chromosome banding patterns in healthy dogs. I got the dead stray dogs from the health care center in Sapporo and obtained bone marrow by sawing the dog legs, which was far from comfortable process. I analyzed the chromosome banding patterns, and this study became the world's first report of the chromosome analysis of the dog. Thereafter, I performed the chromosome analyses on many animals including Clethrionomys rafocanus, bedfordiae, Cervus hortulorum, Japanese crane, gorilla, orangutan, lemur, Indian barking deer, cockroach, harlequin bug, grasshopper, eel and the rest. Many of the studies were merely observational study, although some of them could be reported as a paper. It was the times that a sense of fun was a part of research.
In the meantime, I started also the research of X-inactivation, meiosis in mice with chromosomal translocations, developmental process in sperm and egg with chromosomal abnormalities, with a supervising of Dr. Nobuo Takagi, an assistant professor who had come back from overseas study. We found that although sperm and egg with chromosomal abnormalities had fertilizing capacity at the comparable levels to normal ones, individuals with chromosomal abnormalities died during embryonic development. Through this study, I gained much knowledge in meiosis and developmental process of embryo. These research results were published in Cytogenetics and Cell Genetics, which had a high impact factor at that time. I think even now that this study can be listed up at the top of my studies so far. This is all thanks to kind supervising by Dr. Takagi. Before Prof. Sasaki was killed by pancreas cancer, he told that I was lucky and happy to be supervised by Dr. Takagi. I really hope so. I could learn fun of science and attitude to science from daily talking with him.

Roswell Park Cancer Institute Era
When three years of the my study life passed at Zoological Institute, Hokkaido University, I was supposed to go overseas study to Dr. Avery A. Sandberg Laboratory at Roswell Park Cancer Institute (RPMI) in New York state by the advice of Prof. Sasaki. The laboratory had had a lot of my seniors graduated from Zoological Institute coming to visit over many years, and I was the 13th visitor. Different from general overseas study, the salary was paid by New York State, and my annual income was 13,000 dollars. One dollar was exchanged to 360 yen in those days, and thus my annual income was equivalent to 4,680 thousands yen, the income was much enough to me, a young single man.
When I started overseas study to USA, Philadelphia chromosome, namely t(9;22) observed in chronic myelogenous leukemia (CML) was only chromosome abnormality that was associated with a specific tumor. My research project in the RPMI was to investigate whether such chromosome abnormalities are found in different tumors including other leukemias. A few of my seniors told me that they did not think that such abnormalities would be found. Dr. Sandberg told me in detail about the research theme as follows. The world of science changes with every moment and is renewed timely. The RPMI deals with many patients. Thus, only if I deal with the tumors and I make use of the benefit of the RPMI, I am able to study anything. To be honest, I felt that what he said was too rough, however now I appreciate much that I could study freely. I also think that this experience became a starting point of my free-spirit in research, although I reckon that I tend to try too many things that I think is interesting.
I started the research theme by searching chromosome abnormalities in acute myelogenous leukemia (AML and ALL). Incidentally, RPMI 1640 medium routinely used for cell culture was originally developed for leukemia cells in the RPMI. I became an author of eight and ten papers, in which I was credited as a first author and coauthor, respectively, after three years. Among them, important insights were the identification of t(8;21) in AML and t(4;8) in ALL, and near haploid leukemias. These are now used for diagnosis criteria for leukemias. I feel like having done chromosome analysis of almost 300 leukemia cases. Among chromosome translocations that I found at the RPMI, some translocations were later found to be involved in the activation of oncogenes. As well, the losses of chromosomes frequently observed in leukemias and solid tumors were later found to be involved in the activation
However, curiously, the precise roles of increase of chromosome number have not been clarified so far. I speculated that aneuploidy can cause epigenetic and genetic alteration as a secondary effect to trigger of tumorigenesis, based from the data that I or other researchers have obtained. I really wanted to demonstrate this hypothesis and still so. This desire leaded to my start of development of the new research avenue of medical and biological engineering of chromosome.

Tokyo Medical and Dental College Era
After I spend three years at the RPMI, I became an assistant professor at Medical Research Institute at Tokyo Medical and Dental College thanks to recommendation of Prof. Sasaki at Hokkaido University. I continued to study chromosome abnormalities of mostly (solid) tumors and leukemias for further five years. This was my first experience to live in Tokyo and I was a stranger there. Drs. Shigeo  Mori and Mayumi Mori (his wife) at Department of pathology, University of Tokyo, kindly introduced many researchers who were working in University of Tokyo or graduated from there, and then many collaborative studies started. Dr. Mayumi Mori used to be a colleague in the RPMI. I feel like having done chromosome analysis of almost 300 leukemia cases in Tokyo. I undertook chromosome analysis of all leukemia specimens provided from University of Tokyo, Nihon University, Jichi Medical School, other many universities in Kanto area, Toranomon Hospital, and so. In that time, Dr. Oyashiki Kazuma who had just entered Graduate School of Tokyo Medical University (Present: Professor of the First Department of Internal Medicine, Tokyo Medical University), participated in my study as my first pupil, and worked hard. I knew that there was an immediately perceptible vitality about him. I often drank and talked with him. Around that time, my name had been widely known by many conference presentations and publications.
However, on the other hand, I began to feel uneasiness at the same time. A main cause was the reason why I became famous. In a word, I got surely famous since my name had been credited in many case reports of leukemia, but I thought that it does not necessarily means that I got famous as a scientist and that I might be getting away from true science. I thought that ‘Science’ should be a process in which one interprets various phenomena objectively, makes a hypothesis based on the interpretation, and finally demonstrates the hypothesis. I could not help suppressing emotion that I hoped to carry out the true science but not observational study. I took a decision to carry out true research overseas again. That I felt Tokyo life was not comfortable and that my wife brought up in United States did not get used to Tokyo life also promotes the decision.

In second studying abroad to the United States, there were two candidates. One was University of California, Los Angeles (UCLA), and the other was National Institute of Environmental Health Sciences (NIEHS/NIH) in Research Triangle Park at North Carolina. After many exchanging with them, I decided the latter since research environment and labor condition were better. Regarding research environment, the experimental systems were available to analyze the relationships between aneuploidy and carcinogenesis, which I had thought in a question during chromosome analyses of leukemias.
The group leader was Dr. J. Carl Barrett, an up and coming biochemist and cell biologist. He was two years older than I and had just moved there. He was performing cancer experiments with cell transformation model of Syrian hamster embryo cells in culture (SHE). What he had aimed was to delineate the cause of tumorigenesis by diethylstilbestrol (DES), a hormone or asbestos. His hypothesis was that aneuploid is the mediator of tumorigenesis caused by them. He required cytogeneticists to demonstrate the hypothesis, while I required the experimental systems to analyze the process of tumorigenesis. In other words, we met each other’s needs. Consequently, our study clarified that one of causes of tumorigenesis by DES or asbestos is the induction of aneuploid. Moreover, we clarified that asbestos fiber and DES induces aneuploid by interfering chromosome disjunction (segregation) or by inhibition of centriole formation required for chromosome disjunction, respectively.
Around that time, it was reported that the activation of proto-oncogenes is the cause of tumorigenesis by the experiments based on transfection of DNA prepared from bladder cancer cells into NIH/3T3 cells. It was also reported chromosome translocations mentioned as above were involved in the activation of proto-oncogenes. While the relationship between tumorigenesis and activation of proto-oncogenes was focused, I had another hypothesis that if the activation of oncogenes is sufficient for tumorigenesis, then normal cells should have normal karyotypes after artificial induction of tumorigenesis by oncogenes. However, the truth was not so. We found the loss of specific chromosomes from normal cells after artificial induction of tumorigenesis of normal cells by oncogenes. This suggested that tumorigenesis of SHE cells required the loss of normal chromosome (Chr. 15) as well as the activation of oncogenes. Stated differently, the activation of oncogenes was necessary but not sufficient for tumorigenesis and the loss of normal chromosome(s) or specific genes are also required for it. This became the first experimental evidence to reveal the existence of tumor suppressor genes, and was published in Nature, one of the top journals.
This concept was expected to some extent from fusion experiments between normal cells and cancer cells, and was not the one that suddenly flashed into my mind. It had been known that the fusion cells lost characters of cancer and that these cells gained again characters of cancer with loss of specific chromosomes during successive culture. These reports further let me to have another hypothesis as follows. That is, if the loss of normal chromosomes is necessary for tumorigenesis, reciprocally the introduction of normal chromosomes (genes) to cancer cells might lead to loss of characteristics of cancer. The problem to examine this possibility was how to demonstrate it. At that time, Fournien and Ruddle had reported the method of chromosome transfer at 1977, although it had not been applied to many studies. I thought that this method enables us to know the significance of loss of chromosomes and to identify the chromosomes harboring tumor suppressor genes, by introduction of chromosomes to cancer cells. Unfortunately, the first publication based on this idea was from Stanbridge’s group in United Sates, in which cervical cancer was used, but not from our group. We were left behind them. One should reckon that there are always researchers who are promoting the similar study to anyone’s. That is a universal truth about research. Research has an aspect of game in which speed plays an important role, and thus to have speed is one of abilities.
Around that time, Dr. Makoto Umeda, Professor, Yokohama City University visited at NIEHS/NIH and I had a chance to see him. I had known him, as a grand person of The Japanese Tissue Culture Association, The Japanese Environmental Mutagen Society, and The Japanese Society of Pathology, although it was the first time to see. After I introduced my research to him, he presented a plan to reorganize Kanagawa Medical Center to Kanagawa Cancer Center  and to set up newly a clinical research institute. Furthermore he approached me as my interest in assignment of a laboratory chief of Cytogenetics division. I had not had any particular complaints to NIEHS, and my study in NIEHS was rather going well. In addition, Dr. J Carl Barrett, my host scientist kindly told me to stay as ever, with an offer of permanent position. However, I could not resist my desire to become independent, and there was no choice to go back the same way in my mind.

Kanagawa Cancer Center Era
Finally I decided to go back to Japan and to bring Dr. Minoru Koi, a post-doctoral fellow of NIEHS (Present, in United States) to Japan as a research partner. He went back half a year earlier than I, meanwhile I had to complete research in progress in NIEHS and to make a plan after I go back to Japan. I set a plan to demonstrate the existence of tumor suppressor genes and the identification of them by chromosome transfer experiments, as the theme, as mentioned above. I told Dr. Koi who had already gone back to Japan, about the plan, and then the research started.
First of all, a generation of human monochromosomal library housed in mouse A9 cells was required for chromosome transfer. I forwarded the information that I had collected to Dr Koi, and the project started tentatively. After half a year, I was assigned to a chief in Kanagawa Cancer Center and the project had fully started. Simultaneously, Dr. Hiroyuki Kugoh, an Associate Professor of my present laboratory (Present: my substitute professor since 2014 ) joined to the laboratory as a staff. As long as we once started the project, we decided to generate a library to enable single chromosome transfer of all the chromosomes as a goal. It took two to three years to complete it with many trials and errors. Drs. Koi and Kugoh were responsible for acquisition of hybrid clones containing a single human chromosome, and I was responsible for chromosome analyses of the clones. We could obtain human/mouse A9 hybrid cells that enable chromosome transfer for hChr. 1 and 11 at the early stage. We demonstrated that introduction of hChr.11 could to SiHa cells, a cervical cancer cell line could lead to suppression of its cancer characteristics, and published a paper, in which Dr. Koi was credited as the first author at 1989. To present the data in a conference with the impact as much as possible, we presented the data ranging from the generation of human monochromosomal library to suppression of tumor characteristics by transfer of autosomal chromosome, as a series consisting of five posters. As expected, NHK (Japan's national public broadcasting organization) reported our study with a crowd of people watching our posters .
When I presented data to demonstrate suppression of cancer characteristics by introduction of chromosome after one to two years, Dr. Yoji Ikawa, a grand person of RIKEN asked me a question. In this case, ‘severe criticism’ was more appropriate than ‘question’. His comment was that a new age had began that the researchers should try to clarify the gene function by introduction of a single gene such as oncogene, and that the methodology of chromosome transfer was too rough, nonsense, and it was impossible to identify the tumor suppressive genes, since chromosome carried many and unknown genes. Dr. Ikawa had already been a giant in virology and I now guess that he was trying to interpret life phenomenon from a phenomenological standpoint at the level of a molecule. I immediately argued against his comments. I commented that our method would be able to clarify at least whether a chromosome carries genes involved in carcinogenesis and that our method allowed the physiological gene expression (in the expression level, regulation of expression, and expression of splicing variants). Also I told him that his methodology based on ectopic overexpression of cDNA driven by exogenous promoter was much rougher than chromosome transfer, since the method might not necessary reflect the physiological gene function. After about ten years, I talked Dr. Ikawa about the conversation of that time. Both of us laughed, since we had known by then that both methodology were correct (and complementary to each other).
Around that time, I had an important encounter with two persons. One was Prof. Ken-ichi Matsubara at Institute for Molecular and Cellular Biology, Osaka University (at that time). He told that a human monochromosomal library that we were going to generate was quite important as a resource for human genome research. He offered a support for research fee to complete the library generation. This became a big support for me economically and mentally. The other person was Dr. Yusuke Nakamura at Japanese Foundation for Cancer Research (at that time, Present in The institute of Medical Science, The University of Tokyo, and The University of Chicago). He was an up and coming researcher who had just gone back there from The University of Utah. Incidentally, he used to be a pupil of Dr. Ken-ichi Matsubara.
The mapping of human gene was one of important subjects around that time, and BAC (Bacterial Artificial Chromosome) library containing human DNA fragments were required for the purpose. A human monochromosomal library can be an important material for this BAC library, and thus the library housed in mouse A9 cells that we generated was actually used. I had not considered this kind of application for the human monochromosomal library, and it was a pleasant surprising to know the much wider application of the library than expected. Dr. Yusuke Nakamura thereafter has been playing a quite important role in genome study, in particular diagnostic and therapy for cancer, not only in Japan but also across the world. In that sense, I can say that our research achievements consequently contributed to medicine and health care although we had not directly aimed at them.
 Getting back to our original goal, the identification of tumor suppressor genes by using a human monochromosomal library, this study also went well. We found that there were three patterns for tumor suppression from our studies using tumor cell lines. The first is the induction of cellular senescence in tumor that is originally immortal. The second is the suppression of tumorigenicity in nude mice. The third is the suppression of metastasis without the suppression of tumorigenicity. As for the induction of cellular senescence, we found that hChr.1 carried the responsible gene(s). This study was published in Science (1990) and was spotlighted internationally. Also this study was introduced in ‘Newton’, a Japanese popular scientific magazine.

Tottori University Era
When I was assigned to Professor at Tottori University was April, 1990 when I was 41 years old. In the preceding year, Dr. Hideya Endo, the first head of Medical Institute of Bioregulation, Kyushu University phoned to me in Kanagawa Cancer Center, and told me that he wanted to see me as soon as possible. He was a grand person such as he was designate as a chairperson of the Japanese Cancer Association. A few days later, he came to see me all the way. He presented a plan to newly establish School of Life Sciences, Japan’s first school in Faculty of Medicine to cultivate researchers (but not doctor) to have knowledge of medicine at Tottori University. Then his asked me to be assigned as professor of Department of Molecular and Cell Genetics, School of Life Sciences. As a condition for starting it, I was able to choose and hire an associate professor, an assistant professor, and a technical assistant, as if I could draw a picture on empty canvas in my hands. The only problem was that I would not be able to have a laboratory place for a while, since even the construction of School of Life Sciences Building was still in negotiations with the Ministry of Education, Science and Culture (name at that time). In the long run, I could not have the laboratory space until the beginning of 1994 , three months before the first generation students graduated from the university. I had been borrowing two rooms of Department Molecular Biology that Prof. Endo belonged to (Present; 6th floor of Alesco Building in Faculty of Medicine), until then. As the members of my laboratory increased, I borrowed different rooms temporally.
When I moved to Tottori University, I continued the studies to identify tumor suppressor genes and genes to induce cellular senescence at first. Around that time, Dr. Takashi Sugimura, the Head of National Cancer Center and Dr. Masaaki Terada, the chief of Genetics Division, National Cancer Center supported me by enrolling me to a member of Scientific Research Team on Priority Area (Cancer).  Thank to their help, I could run my laboratory fairly well without fund shortage. What I can still call the scene to mind is that these two persons that many researchers who had gone back to Japan from overseas continued the same study as that of previous laboratory; however I should promote my original study even if it took time to do. These words of wisdom have been my motto in my life as a researcher since then.
When a few years had passed after I move to Tottori University, two persons in Kirin Brewery Co., LTD suddenly visited me one day. They were Dr. Isao Ishida, Head of Central Laboratories for Key Technology (Present: Professor of Teikyo Heisei University) and Dr. Kazuma Tomizuka, a researcher of the same facility (Present: Head of Biologics Research Laboratories , Kyowa Hakko Kirin Co., LTD). Their purpose of visiting was a request for assistance of their project, generation of mice expressing human antibodies. Kirin Brewery Co., LTD had tried diverse ways for introduction of genomic fragments corresponding to human immunoglobulin, such as microinjection for a few years until then, however it seemed that all the trials had ended up in failure. They had been trying to introduce greater than megabase-sized chromosome fragments containing the human immunoglobulin loci into mouse ES cells whose endogenous immunoglobulin loci were inactivated. When I heard the experimental design, I was not sure whether it could be done and felt that there was a major hurdle to overcome. The reason was that it was not clear whether chromosome transfer was applicable to mouse ES cells, or whether, even if that is the case, chimaeric mice containing chromosome fragments could be obtained. I felt unsure since no one had ever tried, on the other side of the coin, I felt excited because of the same reason. Finally I decided to perform the research with them. The biggest problem at the beginning was how we should minimize the size of chromosome fragment containing human immunoglobulin loci.
Tottori University Group provided technical assistance of chromosome transfer to Kirin Brewery group, and then the subsequent study was done in the laboratory of Kirin Brewery in which Dr. Tomizuka acted as a project leader. The review meeting of the results was frequently done both in Tottori University and Kirin Brewery. We started firstly telomere seeding to minimize the size of fragment of hChr. 2 carrying human immunoglobulin light chain locus. Telomere seeding is a technique for fragmentation of chromosomes based on that cloned human telomeric DNA can integrate into mammalian chromosomes and seed the formation of new telomeres. We applied the technique to mouse A9 cells containing hChr. 2, however we could not enough number of clones and the trials had ended up in failure. As a next strategy, we aimed to isolate A9 cells containing the minimum fragment of hChr. 2 carrying human immunoglobulin light chain locus by naturally occurring chromosomal deletion. Fortunately, we could successfully obtain an A9 cell clone containing the minimum fragment of hChr. 2 that carried nearly only immunoglobulin light chain locus located near the centromere and lost other regions. We referred this chromosomal fragment to as W23. Again fortunately, we could obtain mouse ES cells containing W23 via chromosome transfer and found that W23 could be transmitted to the offspring as an independent minichromosome through the germline. We referred mice carrying chromosome (fragments) to as Trans-chromosomic (TC) mice, and this term was listed in The Encyclopedia of contemporary words 1998 (JIYUKOKUMINSHA)
However, in order to generate mice expressing functional human antibody, we needed to introduce chromosomal fragments containing hChr. 14 carrying immunoglobulin heavy chain into TC mice of W23. Thus, we aimed to isolate A9 cells containing the minimum fragment of hChr. 14 carrying human immunoglobulin heavy chain locus by naturally occurring chromosomal deletion. To our surprise again, we could successfully obtain an A9 cell clone containing the minimum fragment of hChr. 14 carrying nearly only immunoglobulin heavy chain locus. We referred this chromosomal fragment to as SC20. Subsequently, we could obtain chimaeric mice containing both W23 and SC20 and found that both of them could be transmitted to the offspring as an independent minichromosome through the germline. The human immunoglobulin heavy chain locus maps at terminal region of long arm of hChr. 14. Thus, SC20 that does not carry other genes was generated by naturally occurring interstitial deletion. Such a lucky thing is nothing less than miraculous! It's not too much to say that this miracle changed the destiny both of Kirin Brewery and Tottori University groups. 
When I think back the reason why this miracle happened around us, there are several reasons as follows. 1) a timely encounter between two groups, 2) our high motivation to breathe new life into chromosome engineering, 3) zeal of Kirin Brewery Co., LTD for new businesses, 4) a brilliant idea of Dr. Ishida and Tomizuka, and the efforts of Dr. Tomizuka to promote the project, 5) my accumulation of knowledge on germ cells, 6) an invisible force that God only knows (the will of God)
We eventually succeeded to generate double TC mice of W23 and SC20 expressing functional human antibody. Furthermore, by crossing the TC mice with mice whose endogenous immunoglobulin heavy and light chain loci were inactivated, we obtained mice with "humanized" immunoglobulin loci. These fruitful results were published in Nature Genetics (Article) and PNAS, pressed in New York Times and Washington Post, and created a sensational headline. Although these results did not create a sensational headline in Japan, the most important thing that I think is that fundamental technology of Tottori University, a regional university was developed to the technology that the world surprises. I think that this development can be attributable to above-described ‘originality’ that we had consistently boosted.
At a later time, it was reported by Dr. Shunichi Takeda at Basel Institute for Immunology, Switzerland (Present: Professor of Kyoto University) that high ratios of targeted to random integration was observed after transfection of genomic DNA constructs in DT40, a chicken pre-B cell line. Since it was also reported by Fonier et al. that targeted integration was observed in human chromosome housed in DT40 cells, we aimed to translocate hChr. 2 to hChr. 14 by this method. We inserted the LoxP site to each chromosome simultaneously housed in DT40 cells by targeted integration, and then induced the translocation between two chromosomes by the Cre recombinase.
Subsequently, we transferred the resultant fused chromosome into mouse ES cells and generated chimaeric mice containing the translocated chromosome. These mice came out later as KM (Kirin–Medarex) mice a commercially available resource to develop antibody-based drugs, jointly by Kirin Brewery Co., LTD and Medarex Inc., a company making antibody-based drugs. The patent right that Tottori University held was assigned to Kirin Brewery Co., LTD.
By the way, I had a right to receive the transfer income, however I did not do so. Instead, I asked Kirin Brewery Co., LTD to establish Department of Human Genome Science, an endowed laboratory in Tottori University for five years with one and a half hundred million yen. This department still exists as a division of School of Life Sciences, Faculty of Medicine, Tottori University by discretion of the university, and plays a part of School of Life Sciences. My conduct like this is based on the aesthetical thought that I refer to as.
The above-described translocated chromosome containing both the human immunoglobulin heavy-chain and light chain loci was later used for generation of TC calves, and I heard that calves for vaccine development are currently under investigation by using the TC calves. Dr. Ian Wilmut, an embryologist who successfully cloned a sheep named Dolly, presented the TC calves expressing functional human antibody as an example that the technique of somatic cell nuclear transfer was effectively utilized. When I read the newspaper in which he talked about somatic cell nuclear transfer and TC calves, I was extremely proud that the technology originating from Tottori University was combined with a global technology such as somatic cell nuclear transfer.
Around that time (October 15, 1996), I was designated as a principal investigator of the research area ‘Life Phenomena’ of Core Research Evolutional Science and Technology Life (CREST) of Japan Science and Technology Agency (JST), and the program for five years started with a budget of seven hundred million yen. When I went to Tokyo for oral examination after passing the paper examination, tension had deprived me of my appetite around the time. I heard later that the competition rate was about 20. Through this experience, I had a strong conviction about that one can be competitive due to his/her uniqueness in the world of research, even if one has no connections, or the institute that one belongs to is regional universities.
The title of the project was ‘Analysis of the regulatory mechanism of genomic imprinting ’. This is a study to aim to identify imprinted genes and to delineate the regulatory mechanism, through comparison of gene expression patterns between paternal and maternal chromosomes by using the human monochromosomal library that we had generated over the years. We identified LIT, an imprinted long non-coding RNA whose size reached up to 50 kb in this study. We further found that LIT maps in imprinting cluster region of hChr. 11 and functions as an imprinting center of this region. It is known now that LIT1 is a responsible gene for Beckwith-Wiedemann Syndrome (BWS), a congenital disease. The research about LIT1 is still carried on by Dr. Kugoh with a focusing on its molecular functions. The study of LIT became a pioneer study to promote non-coding RNA study. We also obtained many new insights in addition to the identification of LIT1. The efforts of Drs. Kohzoh Mitsuya, a first generation student (Present: University of Texas) and Makiko Meguro, a third generation student (Present: Kanazawa University) become one of the main drivers of these studies.
Accumulated knowledge and skills of chromosome engineering was further developed with much diversity. The generation of Human Artificial Chromosome (HAC) is one of the fruitful outcomes. This was generated by modifying hChr. 21 as a starting material. We introduced hChr. 21 into chicken DT40 cells, removed endogenous gene region by telomere-directed chromosome truncation, and introduced a LoxP site as a cloning site of transgene in order to add the function of vector. As well, we could successfully generated Mouse Artificial Chromosome (MAC). These studies were done mainly by Drs. Motonobu Katoh (Present: Assistant Professor, Tottori University), Yasuhiro Kazuki (Present: Assistant Professor, Tottori University), and Masato Takiguchi.
The HAC further gave us an unexpected encounter and a fresh dimension. Starting from encounter with Prof. Shinya Yamanaka, I was again designated as a principal investigator of the research area ‘Fundamental technologies for medicine concerning the generation and regulation of induced pluripotent stem (iPS) cells’ of CREST, JST with a research theme ‘Towards ideal iPS cells for gene therapy and regenerative medicine using a human artificial chromosome (HAC)’ at April 23, 2008.
One of striking achievements in this project was that we could transfer the HAC containing the 2.4 Mb entire human dystrophin gene (DYS-HAC) into iPS cells from Duchenne muscular dystrophy. This study was the first report of complete genetic correction of human dystrophin gene and demonstrated that HAC was a powerful tool for gene and cell therapies. This study was done mainly by Drs. Yasuhiro Kazuki and Masaharu Hiratsuka (Assistant Professor, Tottori University).
After this study was published, domestic and international researchers offered a collaborative study to me. Among them, the collaborative study with Prof. Giulio Cossu in Milano, Italy (Present: London College of Medicine) was very fruitful, which is still in progress, and Dr. Hidetoshi Hoshiya graduated from my laboratory is studying as a post-doctoral fellow of Prof. Cossu’s laboratory. They had started preclinical experiments using blood vessel-associated stem cells (mesoangioblasts) in Italy before then. They succeeded to ameliorate muscular dystrophy in model mice by genetic correction of the mesoangioblasts via introduction of DYS-HAC, followed by injection through arteries.
Furthermore, we demonstrated that HAC carrying Yamanaka factors can be useful for generation of iPS cells. This was achieved by the efforts of Drs. Masaharu Hiratsuka and Yasuhiro Kazuki, and graduate students, Narumi Uno (Present; Assistant Professor, Tottori University)  and Kana Ueda. I expect that it is demonstrated in future that HAC provides safe gene and cell therapies using stem cells.
As a new dimension, the HAC and MAC enabled us to generate model TC mice containing CYP3A cluster (human metabolic genes). This success served as a trigger to start ‘Tottori Bio-frontier project’ in which Tottori prefecture, JST, and Tottori University provide building, equipments, and ground, respectively. As one of the projects, ‘Regional Innovation Cluster Program’ of Ministry of Education, Culture, Sports, Science and Technology and ‘Development of highly sensitive cell systems to monitor chemical toxicity’ of Ministry of Economy, Trade and Industry are progress in the Bio-frontier building.
On a final note, in order to return the results of research to society, Chromocenter (promoter in university: Mitsuo Oshimura) and GPC Laboratory (promoter in university: Dr. Tetsuya Ohbayashi) have been set up, both of which are a Tottori university-launched entrepreneurial venture.

Encounter with Prof. Shinya Yamanaka [Awarded the Nobel Prize for Physiology or Medicine (2012)]
When I listened to his sensational presentation regarding iPS cell generation was the 4th Stem Cell Research Symposium hold in National Institute of Informatics (Tokyo) in May, 2006. I still remember the very good impression that I had then. The study rewrote the book on that terminally differentiated cells never revert to an initial developmental stage. I have been acquainted with him since he was in Nara Institute of Science and Technology in his young days, and I asked him to come see me in Tottori University to discuss a collaborative study and hold a seminar. Prof. Yamanaka and I started the collaborative studies on gene therapy for Duchenne muscular dystrophy by the HAC and safe generation of iPS cells by the HAC, and continuously deepened an interaction with students and researchers of two laboratories. These outcomes were pressed in a prestige newspaper as ‘The world-class iPS technology by Prof. Yamanaka and the HAC technology originated from Tottori University got married.
That is the way I had interacted with him, and thus I felt very happy to hear that he was awarded the Nobel Prize for Physiology or Medicine, as if I was awarded. He has a marvelous personality and has devoted himself to establish a method of treatment of incurable diseases. Although he is still a young investigator, he has a profound sense of humility. He is the way I think the researcher should be. Award of the Nobel Prize to him sends important message to young generation moving away from the sciences. I mean that researchers can contribute to society in a wonderful manner and that basic science is quite important to save many people suffering from incurable diseases. I think that research it will take at least three years  until achievements obtained by Prof. Yamanaka is applied to clinical use. However, it is no doubt that iPS cells is a hopeful technology to profoundly change the future medicine in that various disordered cells can be replace to normal cells. It is noteworthy that iPS cells are useful not only to therapy of well-known diseases such as myocardial infarction and Alzheimer's Disease but also to investigation of the causes and drug discovery for incurable diseases. It really look like that the time of pluripotent stem cells has come. Assertive and loud arguments are apt to prevail rather than essential arguments internationally. However, award of the Nobel Prize to Prof. Yamanaka is a remarkable accomplishment and we, Japanese can argue loudly with confidence that the levels of science and technology of Japan is quite high. I sincerely hope that the encounter and fusion between our HAC technology and the iPS cell technology open up a new avenue for regenerative medicine.

Concluding remarks
The history from chromosome research to medical and biological engineering of chromosome is exactly the history of Mitsuo Oshimura. Needless to say, the achievements mentioned as above are not made by I alone, and these cannot be made without many staffs and students, and homelike atmosphere in the laboratory members. I would like to express to appreciation to all the people that I encountered so far, including mice. All the students and each member’s study are my unforgettable memories. Although I want to present all of them, there is enough space. I would like to do it in from an alternative perspective for another time. If that helps, during the latter stage of the history, I have been just a flagman (director). If medical and biological engineering of chromosome develops further in future, become a world-wide standard technology, and contribute to society like individualization of Tottori University, economic revitalization, health maintenance, and development of human resources, then I would be able to tell that my life as a researcher has meaningful value. On a final note, a network among people continues as an unbroken line, and science is also the case. However, I wish the people of the future to create their own new style, but not to merely drift in the network. Do not be mere followers of me. Treasure encounters and timing. These are my last message to young generations prior to laying down my pen.

Every research has a story and is a matter of deep interest. If possible, I hope to record all of them here, but it is impossible. Therefore, I minimized the list of them to key researches, which will help the readers to understand the path of my research life. For this purpose, among my papers whose number is over four hundreds, I listed up papers that hold a lot of memories for me independent from so called impact factor, with their brief summary. Also, I collected up the press release on newspapers regarding my research since just before I moved to Tottori University, and compiled them as ‘The history of medical and biological engineering of chromosome viewed from press release (written in Japanese)’ and uploaded it to the HP (https://www.med.tottori-u.ac.jp/chromosome ). If one needs further information, please read it.

Oshimura M. et al, J Natl Cancer Inst. 1973 Oct;51(4):1197-203.
Chromosomal banding patterns in primary and transplanted venereal tumors of the dog.
 Venereal tumors of the dog originate from cell transplantation and were spread from one dog to dogs in the world. This is the first paper to report chromosome analysis of dog by differential staining technique.

Sakurai M. et al, Lancet. 1974 Jul 27;2(7874):227-8.
Letter: 8-21 translocation and missing sex chromosomes in acute leukaemia.
This is the second report to identify t(8;21) in leukemias and this translocation later became a diagnostic marker of M2 in AML.

Oshimura M. et al, Cancer. 1976 Aug;38(2):748-61.
Chromosomes and causation of human cancer and leukemia. XVII. Banding studies in acute myeloblastic leukemia (AML).
This is the first report of chromosome analysis in Ph¹-positiveALL.

Oshimura M. et al, Cancer. 1977 Sep;40(3):1161-72
Chromosomes and causation of human cancer and leukemia. XXVI. Binding studies in acute lymphoblastic leukemia (ALL).
This is the first report to identify t(4;8), and this translocation later became a diagnostic marker of ALL and a research object for onset of leukemia.

Oshimura M. et al, Cancer. 1977 Sep;40(3):1143-8.
Chromosomes and causation of human cancer and leukemia. XXIII. Near-haploidy in acute leukemia.
Near-haploidy in acute leukemia is now used for a diagnostic marker of ‘Haploid Leukemia’, an ALL occurred in children, which has a poor prognosis.

Oshimura M. et al, Nature. 1985 Aug 15-21;316(6029):636-9.
Nonrandom loss of chromosome 15 in Syrian hamster tumours induced by v-Ha-ras plus v-myc oncogenes.
This study demonstrated that the loss of tumor suppressive gene(s) is necessary for tumorigenesis in addition to oncogenes such as v-ras plus v-myc, and is the first experimental verification of existence of tumor suppressive genes.

Oshimura M. et al, Cancer Genet Cytogenet. 1989 Apr;38(2):271-82.
Nonrandom karyotypic changes in immortal and tumorigenic Syrian hamster cells induced by diethylstilbestrol.
This study showed that acquisition of tumorigenicity by exposure to asbestos and diethylstilbestrol depends on numerical or structural changes of chromosomes but not mutagenesis.

Sugawara O. et al, Science. 1990 Feb 9;247(4943):707-10.
Induction of cellular senescence in immortalized cells by human chromosome 1.
This study showed that cellular senescence-inducing gene(s) map on hChr.1. . Also this study was introduced in ‘Newton’, a Japanese popular scientific magazine.

Ohmura H. et al, Jpn J Cancer Res. 1995 Oct;86(10):899-904.
Restoration of the cellular senescence program and repression of telomerase by human chromosome 3.
This study is the first report to show that telomerase suppressive gene(s) map on hChr. 3

Tomizuka K. et al, Nat Genet. 1997 Jun;16(2):133-43.
Functional expression and germline transmission of a human chromosome fragment in chimaeric mice.
In this study, we could success to generate mice carrying mini-chromosome expressing functional human antibody. This was the first report of TC mice.

Mitsuya K. et al, Hum Mol Genet. 1999 Jul;8(7):1209-17.
LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids.
We could identify a responsible gene for Beckwith-Wiedemann Syndrome (BWS) by using human monochromosomal hybrids. It was shown later that this gene functions as an imprinting center on hChr. 11.

Meguro M. et al, Nat Genet. 2001 May;28(1):19-20.
A novel maternally expressed gene, ATP10C, encodes a putative aminophospholipid translocase associated with Angelman syndrome.
We identified a novel maternal imprinting gene on hChr.15 and showed that this gene associated with Angelman syndrome.

Shinohara T. et al, Hum Mol Genet. 2001 May 15;10(11):1163-75.
Mice containing a human chromosome 21 model behavioral impairment and cardiac anomalies of Down's syndrome.
We succeeded to generate model mice of Down's syndrome.

Kugoh H. et al, Mol Carcinog. 2002 Nov;35(3):148-56.
Multiple human chromosomes carrying tumor-suppressor functions for the mouse melanoma cell line B16-F10, identified by microcell-mediated chromosome transfer.
This study showed that tumor suppression is mediated by the multiple like cellular senescence induction, suppression of tumorigenicity, and suppression of metastasis, by chromosome transfer.

Kazuki Y. et al, Mol Ther. 2010 Feb;18(2):386-93
Complete genetic correction of ips cells from Duchenne muscular dystrophy.
We succeeded in complete genetic correction of iPS cells from Duchenne muscular dystrophy by using HAC.

Kazuki Y. et al, Gene Ther. 2011 Apr;18(4):384-93
Refined human artificial chromosome vectors for gene therapy and animal transgenesis.
We succeeded to generate HAC with no endogenous genes from hChr. 21

Hiratsuka M. et al, PLoS One. 2011;6(10):e25961.
Integration-free iPS cells engineered using human artificial chromosome vectors.
We succeeded to generate mouse iPS cells by using HAC carrying Yamanaka factors.

Tedesco FS. et al, Sci Transl Med. 2011 Aug 17;3(96):96ra78.
Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy.
We showed that HAC-mediated gene transfer has an efficacy in a preclinical model of DMD and offers potential for future clinical translation.

For readers’ further interest, chromosome transfer technique contributed to 1) identification of DNA-PK as a responsible gene for scid mice, 2) identification of responsible genes for Niemann-Pick disease, Nijmegen breakage syndrome, tumor suppressive genes (KAI-1, TSLC-1), and telomerase suppressor gene (PITX1). Recently, chromosome transfer technique much contributed to generation of model TC mice containing human metabolic genes or model mice for Down’s syndrome.

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