73 The use of epitope-tagged flotillin 1 to identify trophoblast cell secreted extracellular vesicles

Extracellular vesicles (EVs) are composed of lipid-bilayers, are released from cells by outward budding of the cell membrane or through endosomal vesicle fusion with the cell membrane; and they are heterogeneous in size and contents they contain. In humans and rodents, EVs produced by the placenta are present in maternal circulation, and EVs or their contents impact maternal physiology. In contrast to the hemochorial placental structure of human placenta, the cotyledonary synepitheliochorial placental structure of ruminants does not directly contact maternal blood. Furthermore, ruminants lack placental-specific proteins associated with EVs, which would allow for detection and quantification of placental EVs and their contents in maternal circulation. Therefore, methods need to be developed that will allow the detection of placental produced EVs, to determine whether they enter maternal circulation in ruminants. Our goal is to utilise lentivirus-mediated transgenesis to produce epitope-tagged EVs marker proteins in the trophoblast cells of the placenta, to allow for tracking of placental produced EVs in circulation. The protein, flotillin-1 (FLOT-1) is an integral membrane protein thought to be associated with all forms of EVs. The objectives of this study were to (1) identify the presence or absence of bovine FLOT1 in ruminant trophoblast cells, and (2) transduce ruminant trophoblast cells with lentivirus containing a FLOT1-mGFP construct. Bovine tissue-derived RNA was subjected to reverse-transcription polymerase chain reaction detection of bovine FLOT1 mRNA. Amplicons were subjected to gel electrophoresis and dideoxy sequencing. Serum from pregnant cows was used to isolate EVs. A lentivirus vector containing the human FLOT1-mGFP fusion sequence was purchased from OriGene Technologies (Rockville, MD, USA). The FLOT1-mGFP vector, and packaging vectors were used to transfect 293LTV cells using FuGENE 6. Resulting virus was harvested, titered by ELISA for p24 (Cell Biolabs, Inc., San Diego, CA, USA), and used to infect ovine trophoblast cells (oTR). Stably transduced cells were selected by incubation with puromycin. Control oTR cells and stably transduced oTR cells were propagated, media harvested, EVs isolated, and cell lysates generated. Protein lysates and EVs isolated from tissues, cells, serum and media were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, western blotting and immunodetection for FLOT1 and mGFP. FLOT1 was detected by RT–PCR in bovine cotyledon tissue and in cotyledon and serum EVs by immunodetection. The production of a FLOT1-mGFP lentivirus and the transduction of oTR cells were successful. The nontransduced and transduced oTR cells both contained a protein of the predicted size of FLOT1 (~47 kDa); however, the stably transduced oTR cells expressed a larger protein reactive to the FLOT1 and mGFP antibodies (~75 kDa) verifying the expression of the FLOT1-mGFP protein. We now have the reagents in place to stably transduce the trophoblast cells of hatched cattle blastocysts, providing a mechanism to track and identify trophoblast derived EVs.