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In an overwhelming majority of experiments, both mammalian embryonic and somatic cloning have relied on introducing exogenous nuclei into enucleated metaphase II (MII) oocytes. Since attempts at cloning using interphase zygotes as recipient cells have failed, these cells were – until quite recently – commonly regarded as poor recipients for nuclear transfer. However, we have recently shown that interphase zygotes can be successfully used as recipients of embryonic nuclei. In a previous study, we used our original method of selective enucleation (SE), in which the pronuclear envelope with attached chromatin is removed while the liquid pronuclear contents and nucleoli in the zygote’s cytoplasm are left intact, to obtain fertile mice upon transfer of 8-cell (1/8) nuclei into SE zygotes. Here we report that 16-cell (1/16) nuclei can also support full-term development. Additionally, full pre-implantation development, albeit to a limited degree, was obtained after transfer of embryonic stem (ES) cell and foetal fibroblast (FF) nuclei (2.4% and 2.5%, respectively). Sporadically, SE zygotes reconstructed with FF nuclei were able to implant, but they never developed beyond midpregnancy. Our results clearly indicate that SE zygotes can be successfully used as competitive recipients of embryonic nuclei from, at least, the 16-cell (morula) stage. However, their use as recipients of ES cell and somatic cell nuclei seems to be questionable.
Mammalian development is a process, whereby cells from a totipotent zygote gradually lose their potency, i.e. their ability to differentiate, in the environment of the developing embryo. An ideal model for testing the real potential of cells is the experimental production of chimaeras. The first experimentally produced mammalian (murine) chimaeras were created by Tarkowski [1961] and since then many new methods of chimaera production have been developed, including injecting cells into the blastocyst’s cavity or into cleaving embryos. This review describes how different cell types, depending on the developmental stage or culture conditions, manifest their potential to contribute to chimaeras. Cell developmental potential has been analysed in pluripotent blastomeres, which can contribute to all embryonic and extra-embryonic lineages, albeit differently depending on their developmental stage. This is the case in blastocyst lineages, with various developmental potentials depending on the surrounding cells, and in more differentiated cells from different stages of pregnancy, which in some cases may colonise chimaeric animal tissue. Cell potential has also been analysed in embryonic stem and embryonal carcinoma cells, which are pluripotent and efficiently contribute to chimaeras; in multipotent fetal and adult stem cells, which can also participate in chimaera formation; and in somatic mouse embryonic fibroblasts (MEFs), which can also be reprogrammed in the environment of the cleaving embryo. Interspecies chimaera studies have also demonstrated the pluripotency of foreign cells. Experiments with chimaeras have shown that not only pluripotent embryonic cells are capable of contributing to chimaeras, so are adult cells, which plasticity is now well-documented.
This paper reviews the basic knowledge about obtaining farm animal embryos in vitro with special focus on differences among species and application of this procedure in the future. In vitro production of farm animal embryos consists of in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, and in vitro culture (IVC) of embryos. Oocytes can be collected from live animals (by laparotomy, laparoscopy, Ovum Pick Up) or from slaughtered ones (by puncture, sectioning). Usually immature oocytes are isolated, and during IVM they reach maturity. Matured oocytes are cultured with sperm (IVF), leading to the formation of zygotes. In the case of fertilization problems (horse, pig), intracytoplasmic sperm injection is used. The zygotes are usually cultured (IVC) to the morula and blastocyst stages. These embryos can be transferred to recipients or frozen/vitrified. Offspring have been obtained after transfer of cattle, sheep, goat, pig and horse embryos. This procedure can be used in animal breeding, biotechnology, medicine, and basic research.
The objective of this study was to examine the feasibility of identification and selection of cattle embryos based on green fluorescence (GFP-positive) in order to obtain calves carrying an integrated transgene. The construct used (pbLGTNF-EGFP) contained the human tumor necrosis factor alpha (hTNFa) gene fused to the bovine betalactoglobulin promoter (bLG) in plasm id vector pCX-EGFP. In four experiments, 76 zygotes were injected; eight of them developed to the morulac/blastocysts stage of which only five were GFP positive (one of them 100%, one-50%, three- 25%). All of the GFP positive embryos were transferred to recipients. Two calves were born: one after transfer of the 100% GFP positive embryo and the other after transfer of one of the 25% GFP positive embryos. Both animals were healthy with normal weight when compared to two control calves. The integration of pbLGTNF-EGFP in the host genome could not be detected in either of the calves, suggesting that GFP is an unreliable marker for preimplantation screening of embryos.
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