Dr. Hiroki R. Ueda and Dr. Peter Karagiannis
At some point, life can be broken down into components that on their own have no life. Traditional studies have investigated a living body by disassembling it to study the individual components. Synthetic biology takes this approach to its logical next phase, by identifying the appropriate conditions that allow the components to reassemble and reconstitute life. These discoveries have tremendous implications on our understanding of not only the function of the individual components, but also the functions of the dynamics and interactions of the components with each other. The result is better drug development and other medical therapies that have pushed policy and funding to encourage scientists into the field.
Japan's Synthetic Biology Research
Japan has responded accordingly, with a number of actions aimed to create a community for synthetic biology researchers. Most obvious is the founding in 2005 of the Japan Society for Cell Synthesis Research.1 Several years later, Riken, a national research organization with over 15 institutes throughout the country, founded the GenoCon contest, which offers awards for solving iterative synthetic biology problems.2 PRESTO, a national grant agency, has created special funding for synthetic biology projects that seek to design and control cellular functions. In a period of three annual funding cycles beginning in 2010, it has awarded 38 young investigators up to 100 million yen each for 3-5 year projects and given them the opportunity to begin their first independent laboratories.
The abovementioned projects mostly involve synthetic biology up to the cellular level. However, there is a need to shift the field to higher biological tiers like mice to have stronger relevance to human disease and medicines. That is one reason why Riken has partnered with Osaka University to build the Quantitative Biology Center (QBiC). QBiC's ostensible aim is to quantitatively describe biological systems at the cellular level. However, it also has a large synthetic biology team that collaborates closely with quantitative biologists. The expectation of these collaborations is that quantitative theories of the cell will lead to more efficient design of synthetic mice. QBiC represents the strongest relationship yet between Riken and any national university and is testament to the growing importance of synthetic biology in Japanese science policy.
There are a number of steps before achieving synthetic mice, each fraught with errors. At the very beginning is genomic editing, which describes the introduction of foreign genetic material into DNA. The modified DNA is then introduced into stem cells that are injected into the embryos of gestating mothers. There, the cells replicate and grow into the desired mouse type. However, these foreign cells compete with endogenous embryonic cells, often resulting in a litter that is a chimera of the two. The ideal is to produce "100% chimeras" (i.e. mice that contain only the genetic content of the exogenous stem cells; see Figure 1). Although 100% chimeras can be bred, their percentage within a litter is quite low. This inefficiency leads to high costs and significant delays in the acquisition of the mice needed for experiments, hindering both basic and translational research.
Alternative Strategies of Genomic Editing
Most genome editing techniques use the same strategy of introducing double-strand breaks into the DNA and taking advantage of DNA repair mechanisms innate to the cell. Two methods for genomic editing, the TALEN and CRISP/Cas systems, have within the last five years become standard because of their accuracy and easier implementation. Nevertheless, they are still far from optimal. One problem is that the exogenous genes get mismatched, failing to modify the genes as planned. Therefore, even if the exogenous DNA is expressed at satisfactory levels, the mutations are not what the researcher intended, resulting in chimeras of undesired phenotypes. A number of groups in Japan including those at QBiC are working together to build new genomic editing systems based on the above two that both increases the accuracy of the editing and reduces the time to complete it.3
One approach to reducing the number of mismatches in the genome editing and thus undesired phenotypes in the synthetic mice is to increase the size of the exogenous material. Accordingly, several Japanese researchers have considered alternative strategies to TALEN or CRISP/Cas9. One is to iteratively add small DNA fragments into a synthetic vector that can accommodate large amounts of exogenous DNA to the genome.4 This method can clone entire genomes, which reduces the risk of errors and thus mutations. While the short DNA templates used by conventional genome editing methods is a major cause of potential mistakes, they are also easy to prepare. The iterative method exploits this advantage, but because the overall edit is longer than other genome editing methods, the approach should have a smaller fail rate. Another strategy is to substitute the genome completely, something that can be done using human artificial chromosomes (HAC).5 HACs replicate and segregate as though they are natural chromosomes, because they occupy entire genome loci. In other words, unlike the above methods, HACs are not integrated into the existing genome; rather, they compete with it. This technique gives much more stability and reduces common undesirable outcomes like tumorigenecity. Furthermore, for the same reasons, magnitudes more DNA quantity can be introduced. However, neither the iterative strategy nor HACs are easy techniques and thus remain at the experimental stage.
The culturing and selection of the stem cell, neither of which is trivial, also factors into the success of synthetic mouse design. Until recently, stem cells were only derivable from certain mouse strains, which limited the number of disease models that could be studied. Using small molecules that inhibit specific kinases and thus disrupt unwanted differentiation, researchers at Riken were able to breed modified B6 mice, which are standard for most disease models.6 Improvements in the culturing resulted in more than 20% of the litter being 100% chimeras, which improved the success rate by almost a magnitude. Collaborations with several Japanese researchers are aiming to make this success rate even higher.
While the discovery of the above small molecule inhibitors improved culturing conditions, it did not solve the problem of selection. In part because of the variability caused by genomic editing, rarely does a cell culture have a homogenous composition, meaning that cells with the desired genome editing to be implanted into the gestating mother must be separated from the undesired. High-throughput methods like lab-on-chip technology are being developed at various centres across Japan to expedite this task. A great deal of attention is being put into glass materials instead of more commonly used plastic material. Although glass is more expensive, it is also inert to biological substances and thus can be mixed with antibodies that facilitate much more efficient separation.7-8
The field of synthetic biology offers a comprehensive study of biology that calls for scientists of all backgrounds. Japan has recognized this opportunity by committing large amounts of funding and infrastructure that brings together these scientists and also to attract young researchers towards the field. Moreover, the ability to design synthetic mammals like mice will have profound impact on therapeutic medicines and the understanding of disease pathologies. It is for these reasons that Japan views synthetic biology as a major part of its future science policy and aims to rise to the international forefront in the field.
Photo credit: David Robert Bliwas/Flickr/CC
About the Author
Dr. Hiroki R. Ueda graduated with an M.D. from the Faculty of Medicine, the University of Tokyo, in 2000, and obtained his Ph.D in 2004 from the same university. He was appointed Team Leader at RIKEN Center for Developmental Biology (CDB) from April, 2003 and promoted to Project Leader at CDB from September, 2009. In April, 2011, he was made Group Director at RIKEN Quantitative Biology Center (QBiC), where he is responsible for all synthetic biology units. Most recently, he became Professor at the Graduate School of Medicine and Faculty of Medicine, University of Tokyo. He is also one of the founders of the Japanese Society of Cell Synthesis Research.
His primary research interest has been the systems-level study of the network structure of mammalian circadian clocks. These works have led to key findings recently in the underling mechanism of singularity behavior in circadian clocks as well as the temperature insensitivity in mammalian circadian clock. These discoveries have opened the door to diagnostic methods of rhythmic disorders in the body, which has the promise of chronotherapy. Currently, he is using his understanding of circadian clocks to build synthetic biological systems.
Dr. Peter Karagiannis earned his B.S. in 1999 from the University of Toronto and Ph.D. in 2004 from Case Western Reserve University. He came to Japan as a JSPS fellow to study molecular motors at Osaka University. There he transitioned to an educational role until he joined QBiC in 2012 as a science writer. Along with research on molecular motors, he has published on muscle pathologies and models for neural disorders.
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