In response to the expanding landscape of the biotechnology industry and the increasing demand for comprehensive drug development as well as the conduct of preclinical and clinical trials, there is a growing need for employment of diverse animal models, including both small and large animals. The focus of this study was on refining ex vivo culture techniques for bioluminescence imaging following administration of intradermal injections in large animals. To examine the feasibility of our approach, varying concentrations of the rFluc protein were administered to rats and live imaging was employed to validate the corresponding levels of expression. Subsequently, following administration of rFluc to mini-pigs, ex vivo analyses were performed on sample tissues to assess the levels of protein expression across different concentrations. In particular, optimal culturing conditions that facilitated the sustained expression of the protein in samples post-euthanasia were identified. Moreover, by employing small animal imaging devices, we were able to capture clear images of the sample plates, which provided evidence of the successful application of our experimental techniques. The findings from this research represent a significant effort toward refining bioluminescence imaging methods tailored for use with large animal models—an imperative facet of contemporary drug development and biomedical research.

INTRODUCTION
MATERIALS AND METHODS
    Fluc protein expression detection in 96-well plates
    Rat Fluc protein expression (Hulux-2022-07-002, conducted in Hulux animal laboratory,two animals per cage)
    Mini-pig Fluc protein expression (approval and oversight by Endic's Internal EthicsCommittee P233001)
RESULTS
    Enhancement of ex vivo rFluc luminescence by luciferase in an imaging buffer
    Rapid expression of rFluc in small animals in vivo facilitated by the imaging buffer
    Expression conditions for rFluc in large animal skin tissues
    Short-term expression of rFluc according to dose in large animals
DISCUSSION
REFERENCES

• Mina Kim(Department of R&D, Hulux, Seongnam13207, Korea)
• Hao Jin(College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Korea)
• Yong Jin Lee(Department of R&D, Hulux, Seongnam13207, Korea)
• Sang-Myoeng Lee(College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Korea) Corresponding author