25 Rescue of the Hematoendothelial Phenotypes in the ETV2-Null Cloned Pig via Embryo Complementation

Abstract

In transplantation medicine, the embryo complementation method has been introduced as a possible means to produce an organ derived from the desired cells. Previously, our laboratory demonstrated that the transcription factor Ets variant 2 (Etv2) regulates hematoendothelial lineage differentiation. In this study, ETV2-null pig fibroblasts were generated using the CRISPR/Cas9 system, and these cells were utilised as donor cells for porcine somatic cell nuclear transfer (SCNT) to produce mutant embryos. After transplantation of these mutant embryos into 4 surrogate gilts, 12 fetuses were found in 2 gilts at E-18. Eight of those embryos lacked hematoendothelial lineages, were nonviable, and lacked ETV2 by PCR analysis. To rescue the hematoendotheilal phenotypes, the blastomeres were collected from green fluorescent protein (GFP)-expressing embryos, which were generated by SCNT with the pig GFP-fibroblasts. Then, the GFP-blastomeres were injected into the ETV2-null SCNT embryos (4 GFP-blastomeres per a complementation on average) at the morula stage. These complemented embryos were transferred into 4 surrogate gilts. Two surrogate gilts were not pregnant, but 2 pregnant gilts harbored complemented fetuses. Complemented fetuses were evaluated at E26 and had intact and fully complemented hematoendothelial lineages, which were confirmed by CFU-assays, fluorescence-activated cell sorting (FACS), qPCR, and immunohistochemistry. Importantly, the hematoendotheilal lineages completely expressed GFP. In the complemented fetuses, the GFP-positive cells were observed throughout the body at more than 70%. These studies provide a platform for the in vivo production of functional hematoendothelial tissues from pluripotent stem cells (such as human pluripotent cells) using the embryo complementation technique.