Nuclear Transfer in the Mouse
Nuclear transfer (NT) technique provides a powerful experimental tool to study the mechanisms of reprogramming processes and to derive NT-embryonic stem (ntES) cells from living or frozen animals. The Piezo-driven direct microinjection NT method has proved to be a valid technique to clone mice and other species. In addition, this method has been broadly used as a versatile tool for many fields of mouse micromanipulation. This chapter describes the “one step method” protocol of nuclear transfer in mouse, which combines injection of a donor cell nucleus and enucleation of MII metaphase in a single manipulation procedure. This protocol describes the isolation and collection of oocytes, treatment of donor cells, visualization of spindle-chromosomal complex, direct injection and enucleation, activation of reconstructed embryos and their in vitro culture and transfer into pseudopregnant mice.
Key wordsNuclear transfer Microinjection Piezo Cloned mouse Oocytes
Z.L. gratefully acknowledges financial support from the Novartis Research Foundation and the Swiss National Science Foundation (National Research Program NRP63—Stem Cells and Regenerative Medicine) that is provided to the laboratory of Prof. Antoine H.F.M. Peters at the FMI. The authors thank Prof. Antoine. H.F.M. Peters for comments on the manuscript.