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).
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).
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”).
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.
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.
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.
- Dr. 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).
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.
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