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Published on: November 19, 2020
Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei
Shaorong Gao1, Young Gie Chung, Jean W Williams
1The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Abstract:
Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.
Insights
Somatic cell nuclear transfer cloning requires gene silencing for successful development. Cloned embryos retain donor cell traits, impacting their growth and survival in standard culture conditions.
Area of Science:
- Reproductive biology
- Developmental biology
- Genetics
Background:
- Somatic cell nuclear transfer (SCNT) cloning necessitates silencing donor cell genes and activating embryonic genes for successful development.
- Incomplete silencing of donor cell genes can result in abnormal embryonic phenotypes.
- Understanding gene expression dynamics in cloned embryos is crucial for improving cloning efficiency.
Purpose of the Study:
- To investigate the gene expression patterns and culture requirements of cloned mouse embryos derived from myoblast nuclei.
- To determine if donor cell gene silencing is complete during preimplantation development in cloned embryos.
- To compare the characteristics of cloned embryos with tetraploid complementation constructs.
Main Methods:
- SCNT was used to create cloned mouse embryos from myoblast nuclei.
- Cloned embryos were cultured in both standard embryo culture media and somatic cell culture media.
- Gene expression (GLUT4, GLUT1) and glucose uptake were analyzed in cloned embryos.
- Tetraploid complementation constructs were used as a comparison.
Main Results:
- Myoblast-derived cloned embryos thrived in somatic cell media, forming blastocysts with normal morphology and cell allocation.
- These cloned embryos continued expressing the muscle-specific GLUT4 transporter and showed precocious GLUT1 enrichment.
- Both myoblast and cumulus cell cloned embryos exhibited increased glucose uptake, indicating incomplete donor genome silencing.
- Tetraploid constructs displayed a more typical embryonic gene expression pattern.
Conclusions:
- Preimplantation cloned embryos exhibit significant donor cell-type-specific characteristics due to incomplete or progressive silencing of the donor genome.
- Standard embryo culture conditions can disrupt homeostasis and inhibit further reprogramming in cloned embryos, potentially leading to demise.
- Donor cell characteristics profoundly influence cloned embryo development and survival during early stages.
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