Secretory leukocyte protease inhibitor (SLPI), also known as neutrophil elastase and cathepsin-G protease inhibitor, functions in protection of epithelial cells from proteases. SLPI is expressed and secreted by many mucosal tissues, including lungs, seminal vesicles and cervix in women. SLPI plays an important role in protection of endometrial epithelial cells during pregnancy from degradation by degradation by proteases derived from trophoblast at the maternal-conceptus interface. In pigs, SLPI mRNA is known to be expressed in endometrial tissues, but the expression of SLPI in the endometrium throughout the estrous cycle and pregnancy has not been determined. Therefore, we analyzed the expression and regulation of SLPI mRNA in the endometrium throughout the whole stages of the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on Days 0 (day of estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle and on Days 10, 12, 15, 30, 60, 90, and 114 of pregnancy. Real-time RT-PCR analysis showed that the expression of SLPI mRNA in the endometrium increases during midt-o late pregnancy. During the estrous cycle, levels of SLPPI mRNA in estrus and proestrus were higher than those in diestrus and metestrus. In situ hybridization analysis showed that SLPI mRNA was specifically localized to the glandular epithelial cells in the endometrium during pregnancy with strong signal intensity during mid-to late pregnancy. SLPI mRNA was not detectable in conceptus tissues on Days 12 and 15 of pregnancy, but SLPI mRNA was expressed in chorioallantoic tissues during mid-to term pregnancy with increasing levels toward term pregnancy. To determine the effects of steroid hormones, estrogen and progesterone, on the expression of SLPI mRNA, endometrial explant tissues from immature pigs were treated with increasing doses of estradiol-17β (E2) and progesterone (P4). Increasing doses of E2 and P4 increased the expression of SLPI mRNA in endometrial tissues. These results showed that SLPI was expressed in the endometrium in a pregnancy stage-and cell type-specific manner and the expression of SLPI was regulated by E2 and P4 in endometrial tissues, suggesting that SLPI may play an important role in regulating the endometrial epithelial cell function during mid-to late pregnancy in pigs. Further analysis to determine the roles of SLPI at the maternal-conceptus interface is still needed.

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.