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May 28, 2009

Japan Science and Technology Agency (JST)
Central Institute for Experimental Animals
Keio University

Success in Creation of Transgenic Primates

(Enabling research on hard-to-treat illnesses such as Parkinsonís disease)

As part of the Targeted Basic Research Programs of Japan Science and Technology Agency (JST) and of the Strategic Research Program for Brain Sciences of the Japanese Ministry of Education, Culture, Sports, Science and Technology, a group headed by Erika Sasaki, Laboratory Head of the Central Institute of Experimental Animals, and Professor Hideyuki Okano of the Keio University School of Medicine has succeeded in creating transgenic primates. The expression of an introduced gene was discovered not only in a first generation of common marmosets after introduction, but also in a second. This is the first case ever established in the world that an introduced gene was successfully inherited to next generation in primates.
Up until now, the use of mice and rats has played an important role in life science research of transgenic animals (Note 1), but to conduct experimental research on human illnesses, experiments with primates, animals markedly closer to humans functionally and anatomically than rodents, have become necessary. In 1980, the Central Institute for Experimental Animals, which had been working on this problem since the 1970s, succeeded in establishing the common marmoset (Callithrix jacchus ) as a model experimental animal. The marmoset is the smallest animal among the primates and it has a high level of fertility, The Institute has continuously conducted planned propagation of these marmosets as experimental animals.
Using these experimental animals, Professor Okanoís research group at Keio University School of Medicine has made significant progress in regenerative therapy for spinal cord injuries. A group led by Laboratory Head Sasaki, which has been conducting joint research with Professor Okanoís group, has made significant advances in developing model animals for research into human illnesses. This work led to the current success.
In this research into transgenic primates, a foreign gene coding for green fluorescent protein (GFP) was introduced into marmoset embryos in vitro using a viral vector (Note 2). When these embryos were returned to the uterus of surrogate mother marmosets and pregnancy was established, five offspring were derived from four surrogate mothers. All were transgenic. Moreover, it was confirmed that this gene had been incorporated into the reproductive cells of two of the five offspring and a second generation of marmosets was derived from one of these which contained GFP.
Future plans include creating marmoset model animals for human neurodegenerative diseases such as Parkinsonís disease and amyotrophic lateral sclerosis (ALS). Great advances in pre-clinical research can be expected using these models.
The research results outlined here will be published in the May 28, 2009 edition of the science journal Nature.

The achievement described above was principally the result of work on the following projects, research areas and research subjects.
Solution Oriented Research for Science and Technology (SORST)
Research program name : Strategy for Regeneration of the Nervous System by Activating Inherent Neural Stem Cells
Research Spokesperson : Hideyuki Okano, Professor of Physiology, Keio University School of Medicine
Research period : November 2005 to October 2010
For the research subjects cited above, an integrated understanding of three steps was required: (1) the self-renewal and activation of stem cells; (2) the culture of intermediate precursor cells and the regulation of neuroblast migration; and (3) clarification of the formation of a synapse and its maturation in regenerated neurons. To do this there was a conscious attempt to develop visualization techniques, and construct primate models. This work included developing genetic and developmental engineering research methods and an effort to apply them in the interest of making advances in regenerative medicine.

<Background to the Research and its Progress>

The creation of transgenic mice was a major contribution to the field of life science research, particularly the effort to clarify the genetic functions and molecular mechanisms which lead to various pathological conditions. However, in many cases, research results obtained in mice cannot be directly applied to humans because of the many physiological, anatomical and histological differences between mice and humans, which are evolutionarily distinct. For this reason, research using primates as experimental animals that more closely resemble humans in function and anatomy is required. However, there had been no success in generating transgenic primate animals and no scientific proof of the expression of introduced genes in somatic tissue of any derived transgenic primates. In addition, there had been no reports of the transmission of foreign genes to reproductive cells of transgenic primates, which meant life science research using transgenic primates had not been possible.
In that respect, the success of the research group led by Erika Sasaki, Laboratory Head of the Central Institute for Experimental Animals and Professor Hideyuki Okano of Keio University is very important. This group is studying methods of creating transgenic primates that can be used in research in the same way mice are used.

<Content of the Research>

This research group used common marmosets, highly fertile primates, as laboratory animals, which were molded into a uniquely genetically uniform condition with standardized qualities. The many years of work required to do this was headed by Dr. Tatsuji Nomura, Director of the Central Institute for Experimental Animals. The result was the creation of the first five transgenic marmosets. Moreover, they proved that an introduced foreign gene had been transmitted to a next generation of primates. This was the first time this had been done anywhere in the world.
In the creation of this transgenic marmoset, the gene coding for GFP was introduced by injecting a viral vector to the perivitelline alveus of embryos by suspending fertilized embryos in the pronuclear to morula stages in sucrose PB1 media. (See Note 3 and Figure 1a.) The fertilized embryos with the introduced gene were cultured for a few days, and only those fertilized embryos which expressed GFP were selected for implantation in the uterus of surrogate mothers (Figure 1b). The use of this method ensured that all of the five marmosets born would be transgenic (Figure 2), and it was confirmed in the case of four of them that the foreign gene was expressed in various somatic cells. On reaching maturity, the sperm and eggs of two of the five animals were examined, and it was confirmed that the foreign gene was incorporated in their reproductive cells. Moreover, as a result of fertilization in vitro of wild-type marmoset eggs with this sperm, healthy offspring which expressed GFP were obtained (Figure 3).
Injecting the viral vector into the perivitelline alveus using sucrose PB1 media, and selecting the fertilized eggs which expressed GFP for implantation, enabled the efficient creation of marmoset offspring which were 100% transgenic. A foreign gene was expressed in both the somatic cells and reproductive cells of these transgenic marmosets.
Moreover, the use of marmosets already established as experimental animals — animals which among the many primates used in life science research have a relatively short maturation period and are very fertile — made it possible to produce a second generation of transgenic marmosets in only a year and a half.

<Future Developments>

The research group has gained new perspectives on the differentiation and proliferation of self-replicating nerve stem cells and the mechanism of migration of the resultant immature neurons, and it has confirmed various aspects of the basic techniques of developmental engineering in the common marmoset. The creation of the transgenic marmosets was achieved by this group by leveraging an established basic research platform and a dynamically evolving knowledge base.
As the offspring of the transgenic marmosets created are also transgenic and because marmosets are fecund, the performance of replicable experiments using multiple animals in a primate model similar to humans will enable more precise research. We can now expect the use of transgenic primates to result in significant contributions to research on therapeutic methods for hard-to-treat illnesses such as Parkinsonís and ALS. Moreover, as the transgenic marmosets created also express GFP, they may be useful in cell and organ transplant research.
The results achieved in this research assure the progress of Japanese neuroscience research to the next stage.

<Reference Drawings>

Figure 1

Figure 1: Viral vector introduction using sucrose method and rate of expression of the introduced gene

The figure illustrates the introduction of the viral vector to fertilized eggs using sucrose PB1 (sucrose method): (a) Shrinkage of a fertilized egg by inserting it into hypertonic solution, resulting in dehydration of the egg, creating a gap in the transparent zone. When the viral vector is injected into this gap, it enables the injection of more viral vectors to the perivitelline alveus of the fertilized egg. By means of this method, the introduced GFP gene is efficiently introduced and is expressed (b , c).

Figure 2

Figure 2: Five transgenic marmoset offspring are born.

Their names are a: Hisui (Jade), b: Wakaba (Young leaf), c: Banko, d: Kei (left) and Kou (right) : When observed in UV, the skin on the soles of the feet glow green. (In the box in the photo, the foot on the left in the box is that of a wild marmoset).

Figure 3

Figure 3: A second-generation transgenic marmoset

When sperm from a sexually mature transgenic male (Kou, shown in Fig.2d) was used to fertilize eggs of a wild type, the fertilized eggs fluoresced green. Photo at right is of the second-generation transgenic marmoset, named Kouichi (Kouís first son), obtained by implanting the fertilized eggs in the uterus of a surrogate mother.

Figure 4

Figure 4: Photo of a transgenic marmoset on the cover of Nature.

The front cover of the May 28 issue of Nature (provided by courtesy of Nature):
A photograph of a transgenic marmoset has been selected for the cover of Nature. The importance of the achievement is recognized due to the fact that an inserted gene was transmitted to a subsequent generation of transgenic marmosets.

<Explanation of Terminology>

Note 1: Transgenic animals
Animals which enable the elucidation of the function of a gene through the introduction of foreign gene coding for a desired function or by disruption of an inherently present gene.

Note 2: Viral vector
A method involving the use of a virus in which some of the viral genes are replaced by a desired gene so that the pathogenicity of the virus is not expressed while the gene is introduced into representative cells. This utilizes the property in which a virus infects a host and viral genes are incorporated in the chromosomes of the host.

Note 3: Sucrose PB1 media
A media with 0.25 M sucrose (sugar) added. It is used in a method of shrinking a fertilized egg by placing it in a sucrose PB1 solution which is hypertonic to the fertilized egg, resulting in its dehydration. This research uses the properties of the sucrose PB1 media to open a gap between the shrunken fertilized egg and the membrane surrounding it called the transparent body and then injecting the viral vector into that space.

<Title of the Journal Article>

“Generation of transgenic nonhuman primates with germline transmission”
doi: 10.1038/nature08090

<For inquiries regarding this press release>

<About Reseach>
Dr. Hideyuki Okano
Professor, Department of Physiology, Keio University School of Medicine
Tel:+81-3-5363-3746 Fax:+81-3-3357-5445

Dr. Erika Sasaki
Laboratory Head, Laboratory of Applied Developmental Biology,
Marmoset Research Department, Central Institute for Experimental Animals
Tel:+81-44-754-4457 Fax:+81-44-754-4454

<About Japan Science and Technology Agency (JST) >
Dr. Nobuhiro Uchida
Department of Research Promotion, Innovation Headquarters,
Japan Science and Technology Agency (JST)
Sanbancho Building, 3-5, Sanbancho, Chiyoda-ku, Tokyo, 102-0075
Tel:+81-3-3512-3526 Fax:+81-3-3222-2065

<For Media>
Public Relations Division, Japan Science and Technology Agency (JST)
5-3, Yonbancho, Chiyoda-ku, Tokyo 102-8666
Tel:+81-3-5214-8404 Fax:+81-3-5214-8432

Mr. Yoshihiko Kondo or Ms. Nahomi Sato
Public Relations Office, Central Institute for Experimental Animals
Tel:+81-44-754-4457 Fax: 044-754-4454

Ms. Kawagoe or Mr. Kodama
Office of Communications and Public Relations, Keio University
Tel:+81-3-5427-1541 Fax:+81-3-5441-7640


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