This study demonstrated that gene expression in all the blastomeres of an NT embryo is not uniform, and that NT technology by using ear skin fibroblasts can be used to duplicate transgenic pigs without transgene changes.
Ear skin fibroblasts from a transgenic pig expressed eGFP (Fig. 1). After NT using these ear skin fibroblasts, eGFP was detected in 26% of the embryos from the twocell stage to the blastocyst stage (Table 1). More interestingly, among eGFP-expressing embryos, more than half (59%) appeared mosaic; some blastomeres expressed eGFP. but some, which had nuclei, did not express well (Fig. 2). In contrast to the NT-EF group, mosaic expression was not observed in the NT-FF group (Table 1), nor when other groups used eGFP gene transduced somatic cell for porcine NT. To our knowledge, there are no reports of mosaic expression of eGFP or any other gene in NT embryos. The apparent mosaic expression in NT-EF was detected from the two-cell stage to the blastocyst stage.
Nuclear remodeling or reprogramming can be evaluated by studying the change in nuclear structure as well as the expression of specific genes. Winger et al. found that lactate dehydrogenase, citrate synthase, and phospho-fructokinase were all correctly reprogrammed. In another study, it was shown by differential display technology that 95% of the transcripts in NT blastocysts were similar to the control, in vitro-produced embryo. However, that also means that 5% of the transcripts were different. Apparently, Daniels et al. have identified some of this 5%. They show that IL6, FGF4, and FGFr2 are not expressed correctly after NT. Thus, although much remodeling occurs normally after nuclear transfer, in some cases it is not complete.