title: 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.
authors.affiliation: Department of Biomedical Science and Technology, Institute of Biomedical Science & Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea.
authors.forename: Sojung
authors.initials: S
authors.lastname: Kwon
authors.forename: Areum
authors.initials: A
authors.lastname: Kwak
authors.forename: Hyejin
authors.initials: H
authors.lastname: Shin
authors.forename: Soyoung
authors.initials: S
authors.lastname: Choi
authors.forename: Soohyun
authors.initials: S
authors.lastname: Kim
authors.forename: Hyunjung Jade
authors.initials: HJ
authors.lastname: Lim
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D005260:Female; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008297:Male; D049990:Membrane Transport Modulators / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000473:pathology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D036022:Capsid Proteins; C586062:LDP12 peptide; D049990:Membrane Transport Modulators; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C485184:enhanced green fluorescent protein; C108732:methyl-beta-cyclodextrin; D049452:Green Fluorescent Proteins