Somatic cell nuclear transfer in zebrafish

Zebrafish (Danio rerio) have been recognized as one of the best model organisms for the study of developmental biology and human diseases. As of yet, the utilization of zebrafish has not reached its promise. To fulfill this potential, a methodology to generate conditional knock-in/ -out must be developed. Somatic cell nuclear transfer (SCNT) is a potential approach to produce genetically-modified zebrafish. This can be accomplished by transferring gene-targeted cells into enucleated eggs. The primary focus of this dissertation is to improve the efficiency of SCNT. Subsequently, the use of SCNT technology may be extended to enhance the use of zebrafish as a vertebrate animal model. Three important parameters have been characterized and optimized to meet such a goal: recipient eggs, nuclear transfer technique, and cultured donor cells. While the zebrafish cloning technique has been published, it is highly inefficient. Moreover, the existing protocol is difficult to replicate, likely due to poor characterization of zebrafish egg physiology at the time of nuclear transfer. We have demonstrated that, following egg activation, eggs undergo dynamic changes in cell cycle stages and that is likely to affect cloning efficiency. To improve upon this, we implemented a technique in which recipient eggs can be maintained in vitro at metaphase II of meiosis (MII) stage in Chinook salmon ovarian fluid. This should provide a uniform source of recipient eggs for SCNT. Accordingly, we have developed a reliable SCNT protocol that overcomes the challenge of using zebrafish MII eggs with intact chorion as recipient cells for SCNT, by using laser-assisted inactivation of egg genome and micropyle for transfer of the nucleus. This technique has been validated by using phenotypic screening, karyotyping, and genotyping of cloned zebrafish produced. Cloned zebrafish are normal healthy individuals, and go on to produce thousands of healthy offspring. The SCNT technique can be used to produce clones from the major strains of zebrafish used in the research community. Additionally, we have showed that zebrafish SCNT can be used to investigate the influence of donor cell sources on cloning efficiency. By using transgenic fish that express tissue specific green fluorescence protein (GFP) as sources of donor cells, we have found that the type of donor cells used in SCNT influences the developmental capacity of the cloned fish from the blastula stage up to 4 days. In parallel, we have done extensive work to optimize the in vitro culture conditions for zebrafish cells, and described new cell culture and DNA transfection protocols for cultured cells. We explored the possibility of increasing SCNT efficiency by modifying the donor nuclei using histone deacetylase (HDAC) inhibitors. Our SCNT model can be further implemented in combination with existing technology to facilitate gene knock-in/ -out experiments in zebrafish. The ultimate goal is to enhance its prominent role as an animal model for human diseases.