25 Rescue of the Hematoendothelial Phenotypes in the ETV2-Null Cloned Pig via Embryo Complementation
| Autor: | Geunho Maeng, Xiaoyan Pan, Satyabrata Das, Daniel J. Garry, Naoko Koyano-Nakagawa, Mary G. Garry, Kyung-Dal Choi |
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| Rok vydání: | 2018 |
| Předmět: |
Embryo
Embryo culture Reproductive technology Biology Cell biology Transplantation Transgenesis Complementation Endocrinology Reproductive Medicine embryonic structures Genetics Somatic cell nuclear transfer Animal Science and Zoology Induced pluripotent stem cell Molecular Biology Developmental Biology Biotechnology |
| Zdroj: | Reproduction, Fertility and Development. 30:152 |
| ISSN: | 1031-3613 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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