Experimental embryonic-somatic chimaerism in the sheep confirmed by random amplified polymorphic DNA assay

Abstract

Developmental potencies of sheep somatic cells (foetal fibroblasts, FFs) in chimaeric animals were analysed. FFs from pigmented Polish Heatherhead (wrzosowka) breed were microsurgically injected into morulae or blastocysts of white Polish Merino breed (5 cells to each embryo). In one experiment the cells were stained with vital fluorescent dye PKH26, and chimaeric blastocysts were cultured in vitro to confirm the presence of fluorescent cells. In the majority of experiments the blastocysts were transferred to synchronized recipient ewes for development until term. Cultured embryonic cells (CEC), earlier known to produce chimaeras, were injected into blastocysts in control experiments. Seven young were born from FF-injected embryos and three were born from CEC-injected ones. All of them were white, but all three control lambs and three experimental lambs showed small areas of skin pigmentation, which indicated Heatherhead CEC or FF contribution. Tissue samples originating from three germ layers were taken from two FFsoriginating presumably chimaeric lambs (male and female) at the age of one month for DNA analysis. The random amplified polymorphic DNA-PCR method supplied two markers of chimaerism, which were amplification products of 643 bp and 615 bp long DNA fragments, found in tissues of experimental lambs as well as in FFs, but not in the blood of parents of blastocysts. The 643 bp marker was found in the majority of tissues of both lambs. The 615 bp amplicon was detected in the skin and lungs of the female lamb and in the hooves of the male lamb. Our data show that foetal fibroblasts introduced to sheep blastocysts can participate in development and can contribute to all tissue lineages up to at least one month of age.

In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20–24 of gestation

“…This is supported by reports where cells isolated from fetal porcine skin possess a certain level of "stemness" and are able to differentiate into germ-like cells (Dice et al, 2006) and neural progenitors (Zhao et al, 2010). Moreover, fetal fibroblasts from mouse and sheep embryos have contributed to somatic chimeras when injected into blastocysts (Karasiewicz et al, 2008;Piliszek et al, 2007). In comparison with the murine ESC, our putative pig EGC showed an expression pattern slightly directed toward the endo/mesodermal lineage unlike the pFF, which show strong ectodermal differentiation.…”

Animal Biotechnology III

“…Unlike mouse ESCs and EGCs that have been shown to re-enter the germ line in chimeras, pig EGCs have been able to form only chimeras with low percentage of donor-derived cells in the somatic tissues, and have not shown a proven germ line contribution (Shim et al, 1997;Müller et al, 1999, Piedrahita et al, 1998. However, similarly low chimeric contribution has been achieved by injection of somatic cells (fetal fibroblasts) in sheep blastocysts (Karasiewicz et al, 2008) and 8-cell mouse embryos (Piliszek et al, 2007). Therefore, caution is needed when interpreting chimera experiment results, and using proper controls (injection of somatic cells) is necessary to distinguish the "true" stem cells from somatic cells that can be integrated into the embryos after partial reprogramming by the surrounding embryonic cells.…”

Stem cells: Perspectives for the pig in relation to other species

“…While chimeric contribution remains an important standard for pluripotency, some caution in interpreting the results of such assays may be required. This was shown by results from experiments where injections of fetal fibroblasts into mouse or sheep blastocysts have resulted in the formation of chimeras with contribution to all germ layers (Karasiewicz et al 2008, Piliszek et al 2007. When careful analysis was performed, it was determined that the mouse donor fibroblasts had formed hybrids by fusion with the recipient cells (Piliszek et al 2007), which suggests that they may have been reprogrammed in vivo.…”