Optimization Of Nuclear Transfer Protocol And Production Of Transgenic Human Lactoferrin Gene Goat

Somatic cell nuclear transfer (SCNT) so far has been successfully used in producing transgenic animal, making mammary gland bioreactors, and therapeutic cloning etc. To date, although some achievements were reported, the overall cloning efficiency has still remained very low. Optimal SCNT procedures still need to be improved for production of transgenic animals and other purposes. To improve the efficiency of production of transgenic animals using SCNT, this study optimized goat SCNT protocol, and systemically studied the key technical links of production of transgenic animals using SCNT technology.1. Optimization of goat SCNT protocol(1) 1 % ITS was added to oocyte maturation medium (OM) and embryo culture medium (mSOFaa). In results, addition of ITS to OM did not improve the maturation rate, but significantly increased the blastocyst rate of parthenogenesis embryos (P<0.05); addition of ITS to mSOFaa also significantly increased the cleavage rate and blastocyst rate of parthenogenesis embryos (P<0.05). In conclusion, addition of ITS to the medium of oocyte maturation and embryo culture can improve the development of oocyte maturation in vitro and embryo culture; ITS may be applied to free-serum culture of oocytes maturation and embryo in vitro.(2) To improve the electrofusion efficiency of goat SCNT, microelectrode fusion protocol was improved in this study, the microelectrodes whose diameter was 15μm (A), 100μm (B), 200μm (C) were respectively combined and formed A+A, A+B, B+B and C+C. Fusion efficiency from different combinations was evaluated, and pressurization and non-pressurization were compared in each combination. Chamber fusion was control. In results, no significant difference was recorded in the fusion rate of non-pressurized microelectrode fusion groups and chamber fusion group (73.1 % in AA, 75.3 % in AB, 73.30% in BB, 74.2 % in CC, and 74.8 % in CF). The fusion rates in the AA (94.9 %) and AB (92.2 %) groups were significantly higher than that in the AA (83.0 %) and CC (83.9 %) groups. The highest fusion rate and the lowest degeneration rate were obtained in the AA group. These results showed that the pressurized fusion protocol carried out by a pair of tip-end micro-electrodes is optimal to improve fusion efficiency in SCNT. (3) To increase the fusion efficiency of couplets reconstructed with mammary gland epithelial (MGE) cells as donor cells, the effects of phytohemagglutinin (PHA) treatment to couplets reconstructed with MGE cells before electrofusion on the fusion and subsequent development of goat NT embryos was explored in this study. The toxicity of PHA was evaluated by testing its effects on the development of parthenogenetic goat oocytes treated with different doses and durations. The effective dose and duration of PHA treatment (1000μg/mL, 20 min incubation) were selected and used to compare fusion efficiency and embryo development following SCNT. Two different electrofusion protocols, chamber fusion and pressurized microelectrode fusion, were also compared, both without and with PHA treatment (100μg/mL, 20 min). Fusion rate of nuclear donor MGE cells to oocyte recipients was increased from 52.8 % to 74.0 % for chamber fusion protocol (P<0.05) but was not significantly different for microelectrode fusion protocol (72.7 % vs. 78.1 %) after PHA treatment. Fusion rate between both fusion protocols has no significant difference after PHA treatment (74.0 % vs. 78.10 %). There were not significant differences in subsequent cleavage rates and blastocysts development among fused embryos both with and without PHA treatment. The cloning efficiency was significantly improved from 6.7 % to 12.6 % when the recombination of PHA and microelectrode fusion protocol was used. We concluded that the treatment of MGE cells with PHA within a determined range of dose and duration before electrofusion and pressurization has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.2. Establishment of technical system on the multiplication culture of single transfected mammalian cell(4) 96-well cell culture plates were used to isolate cell lineages obtained from a single fetal fibroblast and MGE cell transfected with the pBLM-C1 plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. PCR assays were performed to detect the transfected fragment in positive cells. In results, when a single transfected cell was cultured in 0 %, 50 % and 100 % conditioned medium respectively, the transfected colony rate in 100 % group was significantly increased; the co-culture of single transgenic cell and non-transgenic cells markedly improved the colony rate after multiplication; PCR detection proved that the foreign aim gene has been integrated in transfected colonies. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production. 3. Induced expression and detection of foreign protein from transfected MGE cells(5) The transfected cells were induced by hormone (prolactin + insulin + hydrocortisone) to express human lactoferrin gene. The supernatant was collected 1 time every 6 hours,then was centrifuged for 10～15min (1000 r/min),which was stored in -20℃. The result of western blot analysis on cultured cells supernatant showed that transfected cells can express human lactoferrin protein which the molecular weight is 42 ku.4. Production of transgenic hLF gene goat using optimized SCNT protocol(6) To detect transgenic embryos and obtain viable transgenic goat, this experiment transferred the single transfected fetal fibroblast or mammary gland epithelial cell into the enucleated oocyte using the optimized SCNT technology in above experiments. Reconstructed karyoplast-cytoplast couplets were fused, activated, and cultured in vitro. Partial cloned embryos were used for the dectection of foreign gene, and remaining coned embryos were transferred into synchronized recipients. In results, PCR detection proved that the foreign aim gene has been integrated in cloned embryos. 401 embryos were transferred into 43 recipients, and 11 of them were confirmed pregnancy on 30-45 day by ultrasonography. Regretfully, all of gravid goats aborted within the first 3 months of gestation. In conclusion, though no transgenic goat birth, the technological system using in this study can be used for the production of transgenic goat.