Journal: Stem Cell Reports

Article Title: Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation

doi: 10.1016/j.stemcr.2019.11.008

Figure Lengend Snippet: Apancreatic Phenotype of the PDX1 −/− Fetus and Restoration of Pancreatogenesis by Blastocyst Complementation (A) Apancreatic phenotype of a PDX1 −/− cloned fetus at mid-gestation (day 55; WT, day 66). (B) Chimeric full-term fetuses (three left animals in upper panels) and restored pancreata (lower panels). Fetuses exhibiting huKO fluorescence and male phenotype were determined as chimeras. Chimeric fetuses ( ∗ ) indicating higher level of systemic chimerism showed well-restored pancreata (leftmost in lower panels), while development of pancreatic tissue was poor in a fetus with lower chimerism (center in lower panels). The rightmost animal in upper panels (#) presents a PDX1 -KO fetus showing typical features of the apancreatic phenotype (lower panels). (C) Histological features of the pancreatic tissue generated by blastocyst complementation. Upper panels: H&E-stained sections. Lower panels: sections immunostained for insulin (white), glucagon (green), and huKO (red). Restored pancreatic tissue of the fetus (center) with higher chimerism entirely expressed huKO fluorescence. Scale bars, 100 μm.

Article Snippet: Custom TALEN plasmids targeted to porcine PDX1 , KDR , and HHEX genes, and ZFN plasmids targeted to porcine SALL1 gene, were obtained from ToolGen (Seoul, South Korea) and Sigma-Aldrich, respectively.

Techniques: Clone Assay, Fluorescence, Generated, Staining

Journal: Stem Cell Reports

Article Title: Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation

doi: 10.1016/j.stemcr.2019.11.008

Figure Lengend Snippet: Vasculogenesis-Disabled Phenotype of KDR −/– Fetus and Its Compensation by Blastocyst Complementation (A) A PDX1/KDR dual KO (DKO) fetus with vasculogenesis-disabled phenotype (left) and a chimeric fetus with normalized trait (right) obtained after blastocyst complementation. (B) Left: a PDX1/KDR -KO fetus showing retarded development without blood flow. Center: the appearance of a chimeric fetus showing systemic chimerism with dense distribution of the exogenous huKO-expressing cells. Right: morphology and fluorescence intensity of a normal fetus derived from the huKO transgenic cell. Scale bars, 1 mm. See also .

Article Snippet: Custom TALEN plasmids targeted to porcine PDX1 , KDR , and HHEX genes, and ZFN plasmids targeted to porcine SALL1 gene, were obtained from ToolGen (Seoul, South Korea) and Sigma-Aldrich, respectively.

Techniques: Expressing, Fluorescence, Derivative Assay, Transgenic Assay

Journal: Stem Cell Reports

Article Title: Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation

doi: 10.1016/j.stemcr.2019.11.008

Figure Lengend Snippet: 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).

Article Snippet: Custom TALEN plasmids targeted to porcine PDX1 , KDR , and HHEX genes, and ZFN plasmids targeted to porcine SALL1 gene, were obtained from ToolGen (Seoul, South Korea) and Sigma-Aldrich, respectively.

Techniques: Clone Assay, Immunohistochemical staining, Staining, Derivative Assay, Expressing, Fluorescence, Generated, Marker