We have recently developed a new animal transgenesis method mediated by Tn5 transposase (Suganuma et al. Tn5 Transposase-Mediated Mouse Transgenesis: Biology of Reproduction, published on-line ahead of print August 3, 2005). This technique was successful in facilitating the production of gene-altered animals via intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI), but was not very successful with injections of the Tn5 protein (Tn5p) DNA complex into the cytoplasm or pronuclei of fertilized single cell embryos. As ICSI and ROSI are cumbersome and difficult techniques to master for most laboratories, the failure of simple injections into the cytoplasm to produce transgenic animals was disappointing. We suspect that the failure to integrate the transgene into embryo genomes where pronuclei have formed, is due to the recompaction of sperm chromatin several hours after the onset of fertilization and the formation of membranes around the pronuclei. Therefore, the proposed project will circumvent this problem by involving experiments designed to intercept the sperm chromatin during its decondensation stage soon after in-vitro fertilization (IVF). Thirty minutes after the onset of IVF we will inject the Tn5p:DNA complex (transposome) into fertilized oocytes and follow their development to the late two cell stage. At this stage of development, transcription and translation from the newly formed zygotic nucleus will be detectable. Moreover, the EGFP transgene we will use during these experiments is clearly detectable at the late two cell stage. The transfer of the embryos to surrogate mothers should result in transgenic offspring. This procedure would make transgenesis attempts a reality for many laboratories that find pronuclear microinjection or ICSI difficult. In addition, the presence of a kanamycin gene in the transposome construct used in these experiments will allow the recognition of sites for transgene insertion, and help to determine whether such insertions are random or transposase mediated events. This method will result in a greatly improved rate of transgenesis efficiency. The proposed method will allow easier and more widespread production of transgenic animals. Moreover, it will help the NIH to achieve its mission of fostering an innovative research strategy that may be used to facilitate genetic studies on the causes, diagnosis, prevention, and cure of human diseases. The technique to be developed in this project will enable most academic, agricultural, and pharmaceutical laboratories around the world to produce transgenic animals for a variety of purposes. Transgenic animals may be designed for organ transplants, thereby easing the critical shortage for kidney and liver transplants; they may be used to economically produce proteins or drugs; agricultural and aqua cultural transgenic animals may be implanted with genes to increase disease resistance to improve food quality and supply. Thus, this research fits with NIH's goals to advance the understanding of basic biological systems, improve the control of disease, and enhance health. ? ? ?
