Immunological Genetic Identity among Cloned Dogs with the Same Genetic Background in MHC Class II DLA-DQA1 and DLA-DQB1.

Abstract

The canine major histocompatibility complex (MHC) is dog leukocyte antigens (DLA), which is known to be the most polymorphic genetic system in canine species. Although many cloned dogs have been produced, there was no research about genetic identity of MHC among cloned animals. Recently in Lee's group, two cloned beagles (BG1, 2) were produced by somatic cell nuclear transfer (SCNT) using fetal fibroblast 3 (BF3). Also, four transgenic cloned beagles (Ruppy 1-3, 5) were generated using transgenic BF 3 infected Red fluorescent protein (RFP) gene. We hypothesize that non-transgenic (BG1, 2) and transgenic (Ruppy 1-3, 5) cloned beagles derived from identical donor cell have same genetic characteristic except for RFP gene insertion in immunological site of 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, and Ruppy 1, 2, 3 and 5. 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'-CACCTCGCCGCTGCAACGTG-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 being inserted red fluorescent protein genes 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. This study was financially supported by KOSEF, SNU foundation (Benefactor; RNL BIO), Nestle; Purina PetCare, and the Korean MEST, through the BK21 program for Veterinary Science. (poster)