Figure 3.

Morphological and Histological Features of the PDX1/KDR Dual KO Cloned Fetuses and Its Compensation by Blastocyst Complementation

(A) Immunohistochemical analysis of PDX1/KDR dual KO fetuses and chimeric fetuses with compensated vasculogenesis by blastocyst complementation. Scale bars, 50 μm. Left panels show H&E-stained sagittal section of day 21 fetuses. Top, a PDX1/KDR dual KO (DKO); middle, a chimera obtained by blastocyst complementation; bottom, a non-chimeric fetus derived from the huKO-expressing donor blastomeres. Signals of KDR, PECAM1, and CD34 can be seen (DAB stained) on the vascular endothelial cells of the chimera and donor cell (huKO) derived fetuses. On the other hand, neither vascular structure nor endothelial markers were observed in the tissue of the PDX1/KDR dual KO fetus.

(B) Upper panels: a full-term fetus proven to be chimeric by its phenotypic sex (male) accompanying huKO expression and its restored pancreas entirely expressed huKO fluorescence, indicating that it was generated from the exogenous cells as a result of complementation. Insets show bright-field pictures. Lower panels: the restored pancreatic tissue included well-developed islets stained with α and β cell markers. Scale bars, 100 μm.

(C and D) (C) PECAM1-positive (green) endothelial tissue of a splenic blood vessel in the chimeric fetus exhibiting the donor-cell-derived huKO signal (red). (D) Spleen tissue of the chimeric fetus exhibiting double-positive signals of hematopoietic cell marker (CD45, green) and the donor-cell-derived huKO (red). (C and D) A pair of mirror-image sections were stained due to difficulty of double immunohistochemical staining (lower panels, flipped picture). Scale bars, 10 μm (C) and 50 μm (D).