Sarkar, H.; PRADHAN, B. S.; Batta, S. R.; Wadhwa, N.; Majumdar, S. S.

Mitochondria play an important role in the development of sperm, yet the mechanisms regulating their function in sperm remain poorly understood. The development of a method to regulate the expression of a target gene in the mitochondria of the sperm is an essential step in this line of research. In this study, we attempted to generate a system to express the transgene in the mitochondria of the sperm. As a proof of concept, we generated the transgenic mice expressing green fluorescent protein (GFP) fused with a mitochondrial localization signal (MLS) driven by PGK2 promoter which expresses the transgene in the sperm. Although the PGK2 promoter has been previously demonstrated to drive gene expression in spermatocytes and spermatids, the novelty of our method is the combination of the PGK2-driven MLS-GFP expression to study the mitochondria in vivo. We established two founder lines of transgenic mice by pronuclear microinjection, and MLS-GFP expression was confirmed in the mitochondria of the sperm cells by fluorescence microscopy and flow cytometry. Therefore, we provide a novel platform for studying mitochondrial function in sperm where the GFP can be replaced with other genes of interest to study their effect on mitochondria. These results may also help in understanding mitochondrial inheritance, and mitochondrial dysfunction in vivo.