|EALY, ALAN - UNIVERSITY OF FLORIDA
Submitted to: Cloning and Stem Cells
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2007
Publication Date: 3/21/2007
Citation: Talbot, N.C., Powell, A.M., Camp, M.J., Ealy, A.D. 2007. Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation. In Vitro Cell Developmental Biology Animal. 43(2):59-71.
Interpretive Summary: The presented work reports on the establishment of a cell line collection to be used as a resource by scientist interested in examining the biological changes inherent to nuclear cloning, particularly nuclear cloning in bovine. The majority of cell lines in the collection are trophectoderm cell lines. Trophectoderm is the embryonic tissue which forms the outer layer of the chorion during fetal development and as such establishes the fetal portion of the placenta. Thus, this tissue type is essential for placenta development and function. Since the majority of pregnancy failures in nuclear transfer appear to involve a failure in placenta form and function, trophectoderm cell lines should be a valuable research tool with which to gain understanding of what goes wrong during the establishment of pregnancy of nuclear cloned embryos. The remainder of the cell lines in the collection is of yolk-sac endoderm and embryonic stem cell-derived fibroblast cell lines. These cell lines should also be useful as tools for the study of gene expression changes which occur as a result of the nuclear cloning procedure.
Technical Abstract: Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to better define the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT, in vitro maturation, fertilization, and culture (IVP), or parthenogenetic activation (P). Also, five in vivo fertilized and developed blastocysts were collected by uterine flushing on the 8th day post-fertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVP-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and in many cases interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from parthenogenetic blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.