We previously reported that DNA hypermethylation of SRY promoter is associated with emergence of male-to-female sex reversal. The normality of offspring is achieved by relatively complete and correct nuclear reprogramming during somatic cell nuclear transfer and cloning process. The purpose of this study is to determine whether DNA demethylation of SRY promoter induced by 5-aza-2'-deoxycytidine (AzC) DNA methylation inhibitor may get back phenotypic XY sex reversal female to normal male in SCNT cloning. Canine femoral skin fibroblast cells were established from SCNT-cloned XY sex reversed female (GSF335). Using bisulfite genomic sequencing analysis, DNA methylation levels of SRY promoter in non-treated (normal) and 1uM AzC-treated cells were 88.4% and 55.3% in treatment for 4 days respectively. Seven SCNT-cloned puppies were cloned using the AzC-treated cells as donor cell. Six of those clones showed phenotypically normal male, through one puppy (GSF451) was only observed into male-to-female sex reversal with female genitalia. In umbilical cord tissue, DNA methylation levels on SRY promoter of GSF451 clone and the other clones were 79.2% and 5.7% to 62.2% respectively, which was approximately similar to those of non-treated (normal) and AzC-treated cells. Also, cloned puppies originated from AzC-treated cells implied significantly multiple body weight and height compared to age-matched SCNT-cloned control, which may be underlying in size-effect of AzC-treatment. Our findings suggest that DNA demethylated status of SRY promoter induced by AzC is likely to facilitate normal development including sex differentiation through epigenetic alteration of donor cells.

• Kyu-Chan Hwang(Sooam Biotech Research Foundation)