Inner cell mass-free blastocysts, also referred to as trophoblastic vesicles (TVs) could be used as carriers to be injected with in vitro cultured embryonic cells and to generate clones of animals, providing an alternative method of mammalian cloning. Here we compare three methods of obtaining mouse TVs. Culturing preimplantation embryos in heat shock conditions brings about up to 35% of TVs having up to 47 cells and the efficiency is dependent on the stage of cultured embryos. Live birth was obtained after transfer of ES cell-injected TVs to recipient mice. Culture with radioactive precursor yields up to 55% of TVs, but only at concentrations supplying 37% TVs is their cell number sufficiently high. The effects of PMA treatment are 47% of TVs.
Ovine inner cell masses (ICMs)/embryonic discs cultured in vitro, in conditions copying those in which mouse embryonic stem cells (ESCs) arise from mouse blastocysts, give rise to ectodermal colonies. Day 10-11 ICMs/epiblasts produce ectodermal colonies sufficiently often (55-60%) for it to be considered worthwhile trying to generate presumed ESCs from them. Younger ICMs can only be taken into account if culture conditions can be improved so that ICM/ectodermal cells are more numerous. Older embryonic discs (12-13 day) are inconvenient because of the problem of endoderm overgrowing ectoderm. Secondary cultures of ectodermal colonies form epithelial or mesenchymal cells, which can be passaged at least seven times (50 days).
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