Transgene Expression of Red Fluorescent Protein in Cloned Dogs and Their Offspring.

Abstract

The dogs, Canis familiaris, share many common genetic diseases with human, and therefore have been highly anticipated that dogs would be heavily used for medical research. However, the technical difficulty in getting fertilizable eggs and the unavailability of embryonic stem cells in dogs, has put challenges in generating transgenic dogs. Here, we report the generation of red fluorescent protein (RFP)-expressing transgenic beagles. Canine fetal fibroblasts were stably transfected with a RFP-expressing construct, and used for somatic cell nuclear transfer (SCNT) into enucleated in vivo matured oocytes. Successfully reconstructed 344 reconstructed embryos were transferred to 20 surrogate bitches, from which four female and two male transgenic dogs were born. Tissues from all transgenic puppies were confirmed to carry the RFP transgene. All accessible tissues from live puppies and all tissues (including testis) from a dead puppy exhibited strong red fluorescence. Among them, three female transgenic dogs reached puberty and bred with wild type male dogs by natural mating or artificial insemination. All of the female transgenic dogs were successfully delivered puppies, 9 in total. Five of the puppies carry RFP gene and globally emit red fluorescent light upon UV illumination. In conclusion, SCNT procedure was successfully applied for fertile transgenic dog generation, opening up the greater opportunity to apply transgenic dog models for the study of human diseases. (platform)