| Popis: |
Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE cell epitopes, have been used as antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyper-acute rejection of pig-to-primate xenotransplants. Because GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they are expected to produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs can be used as a novel antibody source for embryo immunosurgery. First, the presence of αGal epitopes in mouse (E3.5), rabbit (Day 4), pig (Day 6–7), and bovine (Day 7–8) blastocysts was examined by staining with fluorescein isothiocyanate (FITC)-conjugated BSI-B4 lectin (Sigma, St. Louis, MO, USA) that binds αGal. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all examined species. Next, pig blastocysts were incubated with a medium containing GTKO pig serum. Swollen TE cells were observed in some of the blastocysts already after 2 min and, after 10 min, almost all TE cells of these blastocysts were completely destroyed. No lysis was recorded when the same experiment was done with wild-type pig serum, suggesting the presence of sufficient quantities of anti-αGal antibodies in GTKO serum to coat the TE cells and induce their complement-mediated lysis. Finally, GTKO serum was systematically tested for immunosurgery. Zona-free blastocysts of the species mentioned above were incubated with heat-inactivated GTKO pig serum for 1 h at 38°C. After washing, the blastocysts were labelled with Hoechst 33342 and TE was stained with FITC-conjugated concanavalin A (ConA) to distinguish the ICM from TE cells. Eventually, the blastocysts were individually incubated in complement solution for 30 to 40 min. Complement-mediated lysis of TE cells was efficiently induced in mouse, rabbit, pig, and bovine blastocysts (10/10, 7/7, 10/10, and 5/6, respectively), and intact ICM were successfully recovered from all species (100, 100, 60, and 80%, respectively). Double fluorescent staining with Hoechst 33342 and ConA clearly showed that the majority of isolated ICM was not contaminated with TE cells. Our study demonstrates that GTKO pig serum is a reliable source of antibodies targeting the αGal epitope of TE cells. Major advantages of using GTKO serum for embryo immunosurgery are (1) that it can be produced easily in large batches, thus reducing experimental variation; and (2) that it reacts with a large number of different species, except for humans, apes, and old world monkeys that lack αGal epitopes. Interesting applications include the preparation of TE and ICM for transcriptome profiling or chimeric embryo complementation experiments. This work is supported by the German Research Council (TR-CRC 127). |
