wait a moment

Mosaic Gene Expression in Nuclear Transfer-Derived Embryos: MATERIALS AND METHODS(4)

Microsatellite Analysis

Genomic DNA from four NT piglets, a donor pig, and the surrogate mother was extracted and subjected to PCR. The microsatellite analysis consisted of three polymorphic porcine loci consisting of different multimers of dinucleotide repeats. The PCR profile included 5 min at 95°C followed by 35 cycles of 30 sec at 94°C, 45 sec at 62°C, and 90 sec at 72°C. PCR products were analyzed simultaneously on 6% polyacrylamide gels on an automatic sequencer using the Genescan and Genotyper software.

Experimental Design

The in vitro developmental ability of the NT embryos derived from ear skin fibroblasts and fetal fibroblasts was compared. After fusion/activation, the reconstructed embryos were cultured for 6 days, examined, and stained in order to count the number of nuclei. In addition, eGFP expression of embryos was examined. The in vivo developmental ability was also determined after transfer to surrogate gilts.

Statistical Analysis

Data were analyzed by ANOVA and the Duncan multiple range test by using general linear models in the Statistical Analysis System program to determine treatment differences. All percentage data were subjected to arcsine transformation before statistical analysis. Data are expressed as mean ± SEM. A probability of P < 0.05 was considered to be statistically significant.