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JABSOM Continues to Blaze the Trail in Gene Technology

JABSOM professor develops new mouse transgensis technique

The first demonstrated transgenesis in mouse.

The first demonstrated transgenesis in mouse

Since Dr. Ryuzo Yanagimachi (Perry, et al., 1999) created the first transgenic mouse via intracytoplasmic sperm injection (famous fluorescent mice), the John A. Burns School of Medicine continues to blaze the trail in gene technology. Recently, Professor Stefan Mosiyadi, from the Institute for Biogensis Research (IBR), has continued this history of gene innovation by developing a new mouse transgensis technique called transposase-enhanced pronuclear microinjection (te-PNI). This method is significant since it yields a greater successful transgenic mouse percentage than the current methods used today.

Throughout the later part of the 20th century and into the 21st century, huge advances in technology and new research discoveries have enabled scientists to manipulate DNA in living organisms to study specific medical conditions.

Scientific Background and Implications

Genes play a vital role in disease and the predisposition for disease. Many disease conditions have a genetic basis. For example, sickle cell anemia, the most common blood disorder in the United States, is a condition which has a clear genetic link due to the mutation of a single gene. Over half a century ago, diseases like this could only be tracked and determined by studying the transmission of the gene through affected offspring. Throughout the later part of the 20th century and into the 21st century, huge advances in technology and new research discoveries have enabled scientists to manipulate DNA in living organisms to study specific medical conditions. One of these advances is “transgenic organisms”, whereby foreign genes are introduced into the host DNA makeup. The hybrid DNA can be created to study and model specific genetic links to human diseases and conditions (Gama Sosa, 2009; Claij, 2006).

Q: What inspired you to a career in molecular genetics?

Prof. Mosiyadi:
My undergraduate degree was in Biochemistry and when I joined CTAHR at the University of Hawaiʻi as a Ph.D candidate in 1986, I intended to pursue the same discipline. However, molecular biology was experiencing a boom at the time and everyone was clamoring for more molecular approaches. Therefore, I switched to molecular biology and when Professor Yanagimachi asked me to take over the transgenesis research at the newly built IBR in 2000, it was a life changing experience for me.

Transgenic Animal Models

Transgenic techniques have been developed in several animals, including insects, fish, birds, rabbits, sheep, pigs, dogs and monkeys. In developing models for human diseases, mice are the most commonly used transgenic model at this time. Mice have many advantages, including low cost, short gestation time, and a well-developed array of technologies and methods for genetic modification. Additionally, since mice are mammals, they share a more similar evolutionary relationship and genome to humans than other low cost animal models, such as fish. This makes them more advantageous since they have a greater potential to model human disease conditions (Gama Sosa 2009, Claij 2006). Common terminology when describing transgenic mice include the term “knockout,” when a foreign gene is introduced to deactivate a specific gene, and “knockin,” which are animals that have a foreign gene added or a native gene enhanced (Charles River Laboratories, 2005).

Associate Professor Stefan Mosiyadi demonstrating the tool used for microinjection.

Associate Professor Stefan Mosiyadi demonstrating the tool used for microinjection

Since the first transgenetic mouse was created in 1974 (Jaenisch & Mintz, 1974), there have been several techniques that have been developed to create transgenetic mice; there are a few basic approaches that are commonly used. These include the following:

  • Pronuclear microinjection (PNI). This is the classical approach where linear genetic material is injected into the pronucleous of the ovum of a mouse. In other words, the new genetic material is injected into a fertilized egg in the single cell stage. This introduces the genetic material into structures known as pronuclei which is the early stage of the embryo nucleus.
  • Intracytoplasmic sperm injection mediated transgensis (ICSI-Tr) is a microinjection technique that uses sperm as the vector for integrating the foreign DNA. The sperm is injected into the egg for fertilization (Perry, et al., 1999).
  • Homologous recombination is a technique that introduces the foreign DNA via genetically modified embryonic stem cells that are injected into the blastocysts. Blatocysts are cells in the mammalian embryonic stage that are ready for implantation in the uterus. This results in chimeric offspring. (“Chimeric” means that a single organism is composed of two or more different DNA cells.)
  • “Viral Systems” introduce the foreign DNA via a deactivated viral vector. This technique has higher gene transfer efficiency, however it is more expensive and has less specificity since viruses tend to splice their genetic payload randomly into the “infected” host cells DNA (in other words, they go “viral”).


    Stephan Mosiyadi (left) with Joel Marh (right)

The new transgenesis method developed by Professor Mosiyadi and his group at IBR, te-PNI technique, was reported recently in the Proceedings of the National Academy of Sciences by Marh et al. The method uses the piggyBac (Urschitz, 2010) transposase gene in the pmhyGENIE-3 plasmid vector with Piezo actuator enhanced pronuclear microinjection to introduce the foreign DNA in a “cut and paste” method. The pmhyGENIE-3 are single construct transposition plasmids which contain both the transposon (the transgene containing component which is transferred to the host DNA) and the self-deactivating transposase (material that is used in the transfer but unimportant afterwards). This procedure was given the acronym of te-PNI which stands for transposase enhanced-pronuclear microinjection. The te-PNI technique has an effectiveness of 25.9% transgenesis efficiency, which is five times more efficient than traditional PNI. Note, the percentage is calculated on the number of total embryos manipulated that are successfully born transgenic. This new method provides a less costly and more flexible cargo capacity of genetic material and yields a higher successful birth rate, compared to traditional PNI.

Q: Tell us about your biotech company, which is a spin-off from this work?

Prof. Mosiyadi
: I am the developer of the pmhyGENIE-3 plasmids and own a small company called Manoa BioSciences which is the first spin-off company formed from research undertaken in the John A. Burns School of Medicine. Manoa BioSciences has signed a licensing agreement with a larger company called Transposagen of Lexington, Kentucky. Transposagen have rights for the worldwide patents of the piggyBac transposase. Manoa BioSciences owns patents for the use of the pmhyGENIE-3 plasmids in many continents, and therefore the licensing agreement between the two companies is very synergistic. Manoa BioSciences has collaborators in China (Dr. Zicong Li), Turkey (Dr. Sema Birler) and Argentina (Dr. Pablo Bosch) for the production of larger transgenic animals as well as several collaborators around the world working with mice. The use of pmhyGENIE plasmids is a new and very recent technological development which will find wider use once it is exposed to the larger scientific community and public by press releases, such as this one, and publications in credible journals from investigators other than the ones in the IBR.

Since the publication by Marh et al., researchers at the IBR have further developed pmhyGENIE-3 plasmids that deliver higher efficiencies to te-PNI in other animal models. This method was recently successful in producing transgenic pigs with a high success rate. The pig transgenesis was achieved by injecting the pmhyGENIE-3 plasmids into the cytoplasm of the embryos. Due to the high lipid content found in pig eggs, the visualization of the pronucleus is impossible, as such, the cytoplasm is the largest constituent of an egg and therefore extremely easy to inject. This is a very simple technique, requiring minimal micromanipulation expertise and allows any laboratory with an inverted microscope and a set of micromanipulators to achieve successful transgenesis. This is a significant achievement since it allows the creation of transgenic animals (i.e. pigs) that represent human disease conditions more closely than mice. This research was done in collaboration with the University of Hawaiʻi alumnus, Dr. Zicong Li, who is currently an Assistant Professor at the Department of Animal Genetics, Breeding and Reproduction, South China Agricultural University, Guangzhou, Guangdong, China.

Q: How did you come up with this method?

Prof. Mosiyadi
: A former PostDoc of mine named Hideaki Yamashiro of Japan and the current IBR transgenic core manager Joel Marh were experimenting in ways to improve and cause a more effective delivery of the pmhyGENIE-3 plasmids into the embryo. When they combined the use of the Piezo actuator employed routinely in cloning and ICSI experiments with the original PNI technique first developed in 1980 they found that the success rate of transgenic incidence improved tremendously. The PNI technique in 1980 had an efficiency of 4.6% by your earlier description, and it had the same efficiency in 2012. Therefore, the introduction of pmhyGENIE-3 plasmids rather than the linear DNA usually utilized and the use of the Piezo actuator and the larger diameter pipettes used in cloning and ICSI made all the difference.

Transgenic Technology used for Medical Treatment

Transgenic technologies have a huge impact on biomedical research, which are translating into clinical therapies. This includes potential development of new strategies to treat and possibly cure human diseases. The results from the use of genetically modified mice have already provided insights on symptoms and treatment for depression and bipolar disorder (Markou, 2010), epilepsy (Chai, 2013), cancer therapies (Wurz, 2013, Masuelli, 2013), and the treatment of diabetes (Absood, 2013). Transgenetic mice can also be used for pharmaceutical research and production, and research in the development of organs suitable for transplant.

Research collaborators at the John A. Burns School of Medicine that are currently using the pmhyGENIE-3 include the following.

  • Dr. Monika Ward, who is trying to produce transfected sperm stem cells, which is extremely difficult to achieve. Her research tries to understanding the function of fertilization for human assisted reproduction.
  • UTMD cavitationDr. Richard Allsopp, who is working in converting adipose stem cells into cardiomyocites for use in patients with myocardial infract (heart attacks). This autologous cell therapy effort will help repair heart tissue damage brought about by the infarct.
  • Dr. Chad Walton, who is trying to develop an in vivo gene therapy in the mouse model by using microbubble technology for the targeted organ delivery of pmhyGENIE-3 plasmids containing a therapeutic transgene in their transposon. This allows gene therapies specific to an organ, without affecting other areas of the body (see image right).

Q: What is your opinion on the current technology and where this will take us in the future?

Prof. Mosiyadi
: Larger species with a closer similarity to the human disease condition are what’s necessary for effective research in cures for diseases and gene therapy. The higher percentage efficiency of the te-PNI technique described within, make it very cost efficient to produce such large animals in the future.

This new technique may have a direct impact on the health and quality of life for people suffering from diseases, such as cancer, diabetes, asthma and mental illness. In addition, the use of animal models can also provide insight to therapies for those carrying the genetic potential, but who have not yet manifested disease. There is no doubt that disease may be caused by several factors and gene interactions, however, the use of transgenic animal models in disease research is a significant component in understanding the factors, causes and potential therapies to treat many diseases.

  • Absood, A., Gandomani, B., Zaki, A., Nasta, V., Michail, A., Habib, P. M. et al. (2013). Insulin therapy for pre-hyperglycemic Beta-cell endoplasmic reticulum crowding. PLoS One, 8, e54351.
  • Chai, X., Munzner, G., Zhao, S., Tinnes, S., Kowalski, J., Haussler, U. et al. (2013). Epilepsy-Induced Motility of Differentiated Neurons. Cereb.Cortex.
  • Charles River Laboratories (2005). Transgenic Animal Science: Principles and Methods. Charles River Laboratories Technical Bulletin, Spring 2005.
  • Gama Sosa, M. A., De, G. R., & Elder, G. A. (2010). Animal transgenesis: an overview. Brain Struct.Funct., 214, 91-109.
  • Jaenisch, R. & Mintz, B. (1974). Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl.Acad.Sci U.S.A, 71, 1250-1254.
  • Marh J, Stoytcheva Z, Urschitz J, Sugawara A, Yamashiro H, Owens JB et al. (2013). Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear mircoinjection transgenesis. PNAS, 109.
  • Markou, A. & Geyer, M. (2010). Animal Models for Mania and Melancholia Studies in Genetically Modified Mice Suggest Novel Mechanisms of Mood Regulation. Biological Psychiatry, 68, 500-502.
  • Masuelli, L., Benvenuto, M., Fantini, M., Marzocchella, L., Sacchetti, P., Di, S. E. et al. (2013). Curcumin induces apoptosis in breast cancer cell lines and delays the growth of mammary tumors in neu transgenic mice. J Biol.Regul.Homeost.Agents, 27, 105-119.
  • Perry, A.C.F., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyota, Y. and Yanagimachi, R. 1999. Mammalian transgenesis by intracytoplasmic sperm injection. Science 284:1180-1183.
  • Urschitz, J., Kawasumi, M., Owens, J., Morozumi, K., Yamashiro, H., Stoytchev, I. et al. (2010). Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc Natl.Acad.Sci U.S.A, 107, 8117-8122.
  • Wurz, G. T., Gutierrez, A. M., Greenberg, B. E., Vang, D. P., Griffey, S. M., Kao, C. J. et al. (2013). Antitumor effects of L-BLP25 Antigen-Specific tumor immunotherapy in a novel human MUC1 transgenic lung cancer mouse model. J Transl.Med, 11, 64.

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One Response to JABSOM Continues to Blaze the Trail in Gene Technology

  1. Truc Nguyen May 31, 2013 at 5:20 am #

    Such cutting edge work going on at UH. Good to read it encapsulated and digestible outside of journals.

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