Submitted to: Cloning
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 2, 2000
Publication Date: N/A
Interpretive Summary: Animals can now be produced from embryos reconstructed from genetically modified fetal and adult cells. Like other types of cloning, this process is plagued by high fetal loss. Significant loss is due to placental abnormalities. In normal development, much of the placenta arises from trophectoderm, the embryonic cell-type surrounding the early embryo. The presence of normal, non-cloned trophectoderm was hypothesized to be able to increase fetal survival by producing a more normal placenta around the cloned fetus. To evaluate protocols to test this hypothesis, a transgene was constructed that allows non-invasive detection of cloned cells within a mixture of cells. By evaluating the expression of a naturally fluorescent protein produced by the transgene in tissue culture, cells can be selected for embryo reconstruction with assured expression of the same transgene in all cells of blastocysts stage embryos. Cloned and normal embryos can then be aggregated to produce a single embryo. Within this aggregate the allocation of cells from the cloned embryo can be assessed by green fluorescence. Techniques were developed to create genetically modified cloned embryos that are surrounded by normal, non-cloned cells. In the process, it was discovered that cells arising from cloned embryos are biased against contribution to the trophectoderm. This technique will allow the original hypothesis that normal trophectoderm surrounding a cloned embryo will increase fetal survival. This technique may allow testing of cloning methods by assay of cell allocation within embryo aggregates.
Technical Abstract: A marker has been developed for detection of blastomeres that originate from embryos produced by nuclear transfer(NT) of genetically modified fetal fibroblasts. This plasmid(phEFnGFP) provides expression in every cell of day 6-8 bovine embryos. Fluorescent and non-fluorescent NT embryos were aggregated on early day 4 and evaluated on day 8. Nuclei of blastomeres that carried the transgene were fluorescent under both UV epifluorescence (Hoechst 33342) and blue epifluorescence (nGFP). There was no bias in the distribution of green fluorescent blastomeres in the ICM or trophectoderm in NT<>NT chimeras. However, there was a strong bias for NT blastomeres to populate the ICM when aggregated with IVP embryos or parthenotes. There was also a strong bias against NT blastomeres when aggregated to IVP embryos. However, the bias against NT blastomeres in the trophectoderm was less (p<0.05) when aggregated with parthenotes as compared to aggregation with IVP embryos. In NT<>NT aggregates, no chimeras were produced that had an ICM composed of blastomeres from a single origin. However in NT<>Parthenote aggregates, 67% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 0% to 83% of the ICM of NT origin. Similarly, in NT<>IVP aggregates 50% of the blatocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 19% to 71% of the ICM being of NT origin. We conclude that production of divaricatead chimeras from NT origin is feasible.