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.

The canine major histocompatibility complex (MHC) is referred to dog leukocyte antigens (DLA), which is known to be the most polymorphic genetic system in canine species. Many cloned dogs have been produced since Snuppy, first cloned dog, there was no research about genetic identity of MHC among cloned animals. Recently in Lee’s group, two non-transgenic cloned beagles (BG1, 2) were produced by somatic cell nuclear transfer (SCNT) using fetal fibroblast (BF). Also, four transgenic cloned beagles (Ruppy 1-3, 5) were generated using transgenic BF transfected with Red fluorescent protein (RFP) gene. We hypothesize that non-transgenic (BG1, 2) and transgenic (Ruppy 1-3, 5) cloned beagles derived from identical donor cells have the same immunological genetic characteristic except for RFP gene insertion in the genome. Thus, the aim of this study is to confirm the immunological identity of DLA class II in cloned beagles produced using same nuclear donor cell. Genomic DNA was extracted from blood of BG1, BG2, Ruppy 1, 2, 3 and 5. Genomic DNA of normal two control beagle, no correlation with BF was also investigated for rulling out the possibility that beagles were inbred. Forward and reverse primers used for DLA-DQA1 and DQB1 respectively were DQAF: 5’-TAAGGTTCTTTTCTCCCTCT-3’ and DQAR: 5’-GGACAGATTCAGTGAAGAGA-3’ DQBR:5’-CTCACTGGCCCGGCTGTCTC-3’ and DQBR: 5’-CACCTCGC CGCTGCAACGTG-3’. Polymerase Chain Reaction (PCR) products were purified, sequenced directly using the Big Dye Terminator kit. Sequencing analysis was performed on an automated 3730xl DNA analyzer. In experiment 1, sequence of DLA-DQ alpha 1 (DQA1) and DLA-DQ beta 1 (DQB1) exon 2, hypervariabel region, was compared in BG1 and BG2. Experiment 2 also compared the sequence of DQA1 and DQB1 among Ruppy 1, 2, 3 and 5. Experimental 3 compared sequence of DQA1 and DQB1 among all cloned dogs (BG1, BG2 and Ruppy 1-3, 5). As a result, BG1 and BG2 have same allele for DQA1 and DQB1 as we expected. They share DQA1*00101 and DQB1*02901 in experiment 1. In experiment 2, Ruppy 1, 2, 3 and 5 also have identical DQA1*00101 and DQB1*02901 allele. No discrimination between transgenic dogs and cloned dogs was seen in DQA1 and DQB1 Allele in experiment 3. DQA1, DQB1 allele was identified as *00101 and *02901 in all dogs. We provided the allele identity of DQA1and DQB1 in cloned beagles, which can be used as preliminary data for immunological related studies. In conclusion, transgenic cloned dogs despite of red fluorescent protein genes being inserted in their nuclear DNA were immunologically compatible with non-transgenic cloned dogs. We demonstrated that cloned beagles produced using identical nuclear donor were immunologically compatible.