Applications for transgenic animal species range from tools in basic research to production agriculture. One major hurdle to improving this technology from its present state is to understand the mechanisms that affect transgene expression. Accurately predicting the transgene expression pattern in a genetically modified animal is vitally important in generating animal models for human disease as well as in designing animal bioreactors for harvesting secreted proteins.
Recent progress in animal cloning by somatic cell nuclear transfer (NT) has allowed researchers to produce animals from genetically defined backgrounds by replacing the genetic material of an unfertilized oocyte with that of a differentiated cell. This technique allows researchers to produce not only multiple genetically identical offspring, but also transgenic offspring by introducing foreign genes into the donor cells before NT. Specifically, progress has recently been made in the cloning of pigs by using somatic cell NT, and recently, genetically manipulated donor cells have been used as donors, which has resulted in transgenic offspring; however, NT technology is still very inefficient.
Nuclear remodeling or reprogramming (or the lack thereof) has been described to some degree in nuclei transferred to enucleated oocytes, but the mechanisms that are responsible for this are still not clearly defined. Although eGFP has previously been used successfully as marker of transgenesis and gene expression, expression has been consistent. In 2000, two transgenic pigs were born that had been derived by injecting an eGFP gene into the per-ivitelline space of a meiotic metaphase II oocyte, which resulted in fertilization, and was followed by embryo transfer. The ear skin fibroblasts from one of those transgenic pigs expressed eGFP. This animal had one copy of the eGFP inserted in to its genome. In the present study, we examined the expression and developmental ability of this eGFP-expressing donor cell line, as well as another eGFP-expressing cell line that was derived from a fetus at Day 35 after NT.