Novel method for demonstrating nuclear contribution in mouse nuclear transfer.
Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.
Munsie
M
M
Centre for Early Human Development, Institute of Reproduction and Development, Monash University, Vic., Australia.
Peura
T
T
Michalska
A
A
Trounson
A
A
Mountford
P
P
D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D002678:Chimera / Q000235:genetics; D005260:Female; D005697:Galactosides; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D007211:Indoles; D007763:Lac Operon; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D013194:Staining and Labeling
D005697:Galactosides; D007211:Indoles; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside