Dog recloning from muscle fibroblasts in transgenic cloned beagle: Regeneration of an identical transgenic dog

Dogs (Canis familiaris) share many common genetic diseases with humans and development of dog disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no recloned transgenic dog has been generated. Here, we attempted the recloning of dogs by nuclear transfer of canine muscle fibroblasts from a transgenic cloned beagle. In order to prepare donor cells for recloning, muscle tissues were collected from a transgenic cloned beagle produced by nuclear transfer of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. We confirmed that the established muscle fibroblasts expressed RFP under an inverted microscope equipped with RFP specific filter (EX 510-560 nm, BA 590 nm). Fused-couplets (178/218, 81.7%) were chemically activated and transferred into the uterine tube of ten naturally estrus-synchronized surrogates. Two surrogates (20%) maintained pregnancy, but only one subsequently delivered one cloned pup. The present study demonstrated the possibility of recloning of transgenic cloned beagles by nuclear transfer.