논문 상세보기
Dog leukocyte antigen identity among cloned dogs with same donor cell
- 언어ENG
- URLhttps://db.koreascholar.com/Article/Detail/354521
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.
