Novel method for demonstrating nuclear contribution in mouse nuclear transfer

Abstract

Confirmation of nuclear contribution is essential to all nuclear transferexperiments. Contribution is easily demonstrated in nuclear transfer progenybut more difficult to confirm in nuclear transfer embryos. The use of donornuclei isolated from lacZ transgenic mice offers a clearand simple method to demonstrate contribution in nuclear transfer embryos andoffspring. The unique line of transgenic mice (Zin40) used in this studydisplays nuclear localised lacZ expression in all cells,including embryonic blastomeres, and demonstrates distinctive blue nuclei whentreated with X-gal substrate. This characteristic staining pattern provided anideal marker for demonstrating nuclear contribution. Nuclear transfer embryoswere generated following serial nuclear transfer of metaphase-arrested nucleifrom transgenic and non-transgenic 4-cell embryos. Totipotency of nucleartransfer blastocysts was confirmed by the generation of live born offspring.Transgenic blastocysts and all tissue samples from fetuses and pups generatedby nuclear transfer displayed distinctive blue nuclei when stained with X-gal.This staining pattern was characteristic of the transgenic mice from which thedonor nuclei were isolated and clearly confirmed nuclear origin. The use ofthis marker will also allow the opportunity to investigate the developmentalpotential of nuclear transfer embryos by examining the contribution of nucleartransfer embryonic cells in chimaeric embryos.