Incorporation of cultured embryonic cells into transgenic and chimeric, porcine fetuses.

Abstract

The derivation of murine, embryonic stem cells, and their use in the generation of transgenic mice, are well-established procedures. Application of these methodologies to non-murine species, however, remains to be fully realized. Non-murine embryonic stem cells would be of considerable value to studies in comparative development, and would raise important implications for agriculture and biotechnology. Here we report the achievement of chimerism and transgenesis in the domestic pig (Sus scrofa) at a fetal stage by the embryonic stem-cell methodology, using an embryonic cell line which previously we described as having the capacity to differentiate in vitro. This entailed: transformation, by electroporation, of a cell line, PE1, with the bacterial gene for neomycin-resistance; re-introduction of a polyclonal population of neo-PE1 cells into host blastocysts, followed by transfer of reconstructed embryos into a pseudopregnant recipient; and subsequent derivation of transgenic and chimeric porcine fetuses, as determined by two independent molecular assays of fetal genomic DNA. In the first instance, chimerism was revealed in one fetus by the presence of the transgene, as detected by Southern blotting; and in the second instance, in that and another fetus by the presence of supernumerary alleles for the class II major histocompatibility locus, SLA-DQB*C, by single-strand conformation polymorphic analysis. The contribution of neo-PE1 cells to the first chimeric and transgenic fetus was approximately 25%, and to the second chimeric fetus, below the level of detection by Southern blotting (i.e. less than 10%). The results indicate that, at the time of embryo reconstitution, a proportion of neo-PE1 cells were pluripotent and of the primary ectodermal lineage.