Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Abstract

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12–EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12–EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12–EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP–LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP–LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.