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

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 FFs-originating 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.

Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology.

The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds were chimeras. The effect of donor cells on the reproduction and physiology of the recipients was evident.