331 BLOOD ANTIGEN-COMPATIBILITY BETWEEN CLONED BEAGLES AND TRANSGENIC CLONED BEAGLES

Abstract

It is known that cloned animals produced by somatic cell nuclear transfer (SCNT) using the same donor cells show immunological compatibility for tissue transplantation. However, immunological compatibility for tissue transplantation or blood transfusion between cloned animals and transgenic cloned animals originating from the same donor cell has not been assessed until now. The objective of this study was to evaluate the compatibility of blood group antigens in cloned dogs with each other, in cloned dogs and transgenic cloned dogs, and in transgenic cloned dogs with each other by a crossmatching test. In Lee’s group, 2 cloned beagles (BG1, 2) were produced from fetal fibroblasts (BF3) using SCNT. Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, although with the transfection of the red fluorescent protein (RFP) gene. All 6 beagles shared the same genetic background except for RFP gene insertion in their genome. Canine blood antigen is labelled as dog erythrocyte antigen (DEA), a critical factor for determining blood antigen compatibility during a crossmatching test. Blood samples were collected from all tested dogs. Serum and red blood cells (RBC) were separated; RBC of all dogs were washed 3 times with 0.9% saline, and a 4% RBC suspension was made from the washed cells. They were mixed in the following experimental designs. In experiment 1, an RBC suspension of cloned beagles was combined with equal volumes of another cloned beagle’s serum. In experiment 2, an RBC suspension of Ruppy 1–3, 5 was mixed with an equal volume of another transgenic cloned beagle’s serum; the reverse reaction was also performed. In experiment 3, an RBC suspension of cloned beagles was mixed with the serum of transgenic cloned beagles and the reverse reaction was performed. All the mixtures were incubated at 37°C for 20 min, centrifuged, and then assessed for hemolysis or agglutination. In experiments 1 and 2, no samples showed any evidence of hemolysis. However, in contrast to the other experiments, experiment 3 showed a different pattern. Although the RBC of transgenic cloned dogs showed hemolysis when mixed with the serum of cloned dogs, the RBC of cloned dogs did not react with the serum of transgenic cloned dogs. From the results, we see that the 4 transgenic cloned beagles could not be donors for blood transfusion, but the 2 cloned dogs could be universal donors for all. In conclusion, cloned beagles and transgenic cloned beagles show blood antigen compatibility within themselves. However, the 4 transgenic cloned beagles showed blood antigen incompatibility with the 2 cloned beagles.