Figure 1.
Generalized scheme for the production of chimeric porcine blastocysts and fetuses by the aggregation method.
For in vitro analysis of the chimeric blastocyst formation, donor ICMs were isolated from parthenogenetic blastocysts derived from IVM oocytes. Isolated ICMs stained with DiI were aggregated with blastomeres isolated from parthenogenetic host embryos in a microwell made on the bottom of a culture dish. For in vivo analysis of chimeric fetus formation, the donor ICMs were isolated from blastocysts fertilized in vitro by transgenic boar sperm carrying the fluorescent huKO gene. ICMs of the IVF blastocysts were similarly aggregated with the parthenogenetic host embryos as the DiI-stained ICMs, and the resultant blastocysts were transferred to recipient pigs to obtain chimeric fetuses.
Figure 2.
Production of chimeric blastocysts with donor ICM and parthenogenetic host embryos.
(A, D) A donor ICM (stained with Dil) aggregated with host blastomeres isolated from parthenogenetic embryos at the morula (A) or 4–8 cell stage (D). (B, E) Bright field images of chimeric blastocysts developed from the aggregated embryos. (C, F) Confocal fluorescence images of chimeric blastocysts showing DiI fluorescence in ICMs. Single confocal sections of fluorescence were overlaid on the bright field images. (G-I) Parthenogenetic host morulae injected with DiI-stained donor ICM (G) and resultant chimeric blastocysts (H, I). Arrow heads, ICM. Scale bars = 50 µm.
Figure 3.
Production of chimeric blastocysts by blastomere aggregation.
(A, D, G) Aggregation of donor (DiI-stained) and host blastomeres between synchronous (A, D) and asynchronous (G) embryonic stages. (B, E, H) Chimeric blastocysts developed from the aggregated blastomeres. (C, F, I) Confocal fluorescence images of the chimeric blastocysts showing DiI fluorescence in ICMs. Single confocal sections of fluorescence were overlaid on the bright field images. Arrow heads, ICM. Scale bars = 50 µm.
Table 1.
In vitro development of the chimeric embryos produced by injection or aggregation method.
Figure 4.
Chimeric fetuses produced by aggregation of the ICM carrying huKO transgene and parthenogenetic host embryos.
(A, B) Morphological appearance of the chimeric blastocysts before embryo transfer. (C–K) Chimeric fetuses (day 18) showing huKO fluorescence derived from the donor ICM cells (C, D, F, G, I, J) and immunohistochemical images showing proportion of the donor-derived (huKO-positive) cells in the tissue of chimeric fetuses (E, H, K). (L, M, N) A day-19 fetus developed from an embryo fertilized in vitro with the huKO transgenic boar sperm as a positive control, showing the systemic expression of huKO (M, N). (O, P, Q) A non-chimeric fetus (day 22) developed from the aggregates of two parthenogenetic embryos as a negative control.
Table 2.
Development of the aggregated embryos into chimeric fetuses.