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Canine induced Pluripotent Stem Cells (iPSCs) can provide great potential for regenerative veterinary medicine and may assist in the development of new therapeutics and pre-clinical studies for both dogs and human. To date, there have been several reports on the generation of canine iPSCs using retroviral or lentiviral transduction of Yamanaka’s factors. However, there is no report of canine iPSCs generated by genomic integration-free methods. According to previous studies, a polycistronic and synthetic self-replicating RNA system was developed for generating human iPSCs by the RNA replicon of Venezuelan Equine Encephalitis (VEE) virus. The VEE replicon is a positive-sense and single-stranded RNA which is similar with cellular mRNA containing a 5’ cap and poly (A) tail. It is no potential for genomic DNA integration problems because it does not use a DNA intermediate. Here, we investigated to generate canine iPSCs by a single transfection of the VEE-reprogramming factor (VEE-RF) RNA. To generate integration- free canine iPSCs, the VEE-OKS-iG RNA that expresses four reprogramming ORFs (hOct4, hKlf4, hSox2 and hGlis1) was transfected. Also, B18R mRNA was co-transfected to reduce immune response by VEE replicon. Putative canine iPSC colonies first appeared between day 15-25. Interestingly, they have two distinct types of initial canine iPSC colonies. They were identified by immunohistochemistry of live cells using TRA-1-60 antibody and also showed clear alkaline phosphatase (AP) activity. Further study will be required to analyze the characterization for a clinical application, and the non-integrating and self-replicating VEE RNA replicon system has the potential to make a great contribution to generating clinically applicable canine iPSCs.