Transgenic animals have been used for years to study gene function, produce 
important proteins, and generate models for the study of human diseases. 
However, inheritance and expression instability of the transgene in transgenic 
animals is a major limitation. Copy number and promoter methylation are known to 
regulate gene expression, but no report has systematically examined their effect 
on transgene expression. In the study, we generated two transgenic pigs by 
somatic cell nuclear transfer (SCNT) that express green fluorescent protein 
(GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and 
bisulfite sequencing were performed to determine transgene copy number and 
promoter methylation level. The correlation of transgene expression with copy 
number and promoter methylation was analyzed in individual development, 
fibroblast cells, various tissues, and offspring of the transgenic pigs. Our 
results demonstrate that transgene expression is associated with copy number and 
CMV promoter methylation in transgenic pigs.