| Breeding and application of new genetically modified varieties of Arbas Cashmere goats with a high economic value is strategically significant for modern animal husbandry. Currently, the main existing technology for breeding new transgenic cloned varieties is transgenic cloning. At present, fibroblasts are predominantly used as nuclear donor cells in Cashmere goat transgenic cloning; these cells have the significant disadvantages of limited number of passages and low survival rate after transfection. Such disadvantages have become a bottleneck in breeding new transgenic varieties of Cashmere goats. Therefore, our study aimed to product transgenic cloned Arbas Cashmere Goats using of Arbas Cashmere ADSCs, BMSCs and MDSCs as nuclear donors for somatic cell nuclear transfer (SCNT). The results of this study should provide experimental basis for the production of transgenic cloned Arbas Cashmere Goats. 1. Establishment of lines of ADSCs, BMSCs and MDSCs of Arbas Cashmere goat(1) ADSCs isolation, culture and identificationADSCs isolation was used collagenase type I and surface markers were identified immunohistochemically. The ADSCs was differentiated into neurocytes, myotube cell and insulin-producing cells and the expression of neuron-specific enolase (NSE), fast muscle myosin and insulin was identified by immunohistochemistry. The results showed that proliferation of ADSCs was stable and was stained positively for vimentin, CD49d, CD44and CD13, but stained negatively for CD34and CD106. Expression of NSE, fast muscle myosin and insulin by ADSCs was strongly induced. The results show that ADSCs own the ability to three germ layers of cells.(2) BMSCs isolation, culture and identificationBMSCs isolation was used mechanical method and surface markers were identified immunohistochemically. The BMSCs were differentiated into neurocytes and osteoblasts and the expression of NSE and osteocalcin was identified. The findings showed that BMSCs were isolated and amplified to express CD13, CD29, CD44and CD73through adherent culture, with no marked signs of aging after multiple passages. Expression of NSE and osteocalcin by BMSCs was strongly induced by neuronal and osteogenic differentiation.(3) MDSCs isolation, culture and identification MDSCs isolation was used twice enzymatic digestion and surface markers were identified immunohistochemically. The MDSCs was differentiated into neurocytes, myotube cells and insulin-producing cells and the expression of NSE, fast muscle myosin and insulin was identified. The results showed that proliferation of MDSCs was fast and was stained positively for Desmin, a-Sarcomeric Actinin, MyoD1, Myf5and PAX7. Expression of NSE, fast muscle myosin and insulin by MDSCs was strongly induced. The results show that MDSCs own the ability to three germ layers of cells.2. Analysis of the sternness of ADSCs, BMSCs and MDSCs from Arbas white Cashmere goat(1) We aimed to explore these similarities further by examining the expression of the pluripotency and sternness biomarkers, AKP, IL-6, Nanog, Oct-4, Rex-1, Sox-2and TERT, as well as the triploblastic biomarkers, Sox-1, Myod1and Gata-6in ADSCs, BMSCs and MDSCs. Immunocytochemistry showed that AKP, IL-6, Nanog, Oct-4, Rex-1and TERT were expressed in ADSCs, BMSCs and MDSCs, whereas Sox-2was not. In ADSCs, the expression of IL-6mRNA was relatively high, followed by Nanog and Oct-4, while Rex-1and TERT expression were the lowest (P<0.01). In BMSCs, the expression of Rex-1was relatively high, followed by IL-6, while Oct-4, Nanog and TERT were comparatively low (p<0.01). In MDSCs, the expression of IL-6, Nanog and Oct-4were relatively high, while TERT was comparatively low (P<0.01). However, no expression of Sox-2mRNA was detected in any of the three cell lines. The expression of Sox-1, Myodl and Gata-6was observed to different degrees in all three cell lines (P<0.01); the expression pattern in MDSCs was different from that in ADSCs and BMSCs. Western-blotting indicated that no expression of Sox-2and Rex-1protein occurred in ADSCs, BMSCs and MDSCs, while the other five proteins were all expressed to different degrees (P<0.01); the expression pattern was consistent with the mRNA results. Of these, MDSCs were found to exhibit the most ESC-like properties and would make the best candidates for seed cells.(2) No teratoma tissue or triploblastic differentiation appendages were formed in NOD-SCID mice after injection of ADSCs, BMSCs and MDSCs. Our results suggest that ADSCs, BMSCs and MDSCs from Arbas white Cashmere goat are non-oncogenic3. Production and detection of transgenic Arbas white Cashmere goats using ADSCs, BMSCs and MDSCs from Arbas white Cashmere goat as nuclear donors for SCNT(1) Transfection and detection of pluripotencyWe used FFCs as control and pCDsRed2-1as an exogenous vector. The ADSCs, BMSCs, MDSCs and FFCs were cultured for transfection respectively. Differentiation was induced in ADSCs-pCDsRed2-1, BMSCs-pCDsRed2-1and MDSCs-pCDsRed2-1as described about. The results showed that No significant differences in the transient transfection efficiencies of four kinds of cells were detected48h after transfection. The rate of neomycin resistant monoclonal colony formation and red fluorescence in ADSCs-pCDsRed2-1, BMSCs-pCDsRed2-1, MDSCs-pCDsRed2-1and FFCs-pCDsRed2-1were78.26%,61.05%,85.40%and50.00%respectively (P<0.01). Expression of NSE, fast muscle myosin and insulin by ADSCs-pCDsRed2-1and MDSCs-pCDsRed2-1was strongly induced, while the integrated exogenous genes did not influence pluripotency as the same as BMSCs-pCDsRed2-1expressed NSE and osteocalcin induced by neuronal and osteogenic differentiation.(2) Production and detection of transgenic cloned embryosUsing of ADSCs-pCDsRed2-1, BMSCs-pCDsRed2-1, MDSCs-pCDsRed2-1and FFCs-pCDsRed2-1as nuclear donors for SCNT to product transgenic cloned embryos. Expression of the DsRed2gene in the four kinds of cells original embryos was investigated at the morula stage using Q-PCR and Western-blot analysis. The results showed that the convergence rates of cloned embryos derived from ADSCs-pCDsRed2-1, BMSCs-pCDsRed2-1and MDSCs-pCDsRed2-1were higher than cloned embryos derived from FFCs-pCDsRed2-1, while their8-cell and blastocyst rates were similar. At the same time, convergence rates of cloned embryos derived from MDSCs-pCDsRed2-1was highest in all four of those. In mRNA level DsRed2-1expression of cloned embryos derived from ADSCs-pCDsRed2-1, BMSCs-pCDsRed2-1and MDSCs-pCDsRed2-1were more than2times greater than that of embryos derived from FFCs-pCDsRed2-1(P<0.01). Similarly, in protein level DsRed2protein expression of cloned embryos derived from BMSCs-pCDsRed2-1was1.29greater than that of embryos derived from FFCs-pCDsRed2-1(P<0.01). Differently, DsRed2protein expression of cloned embryos derived from FFCs-pCDsRed2-1was2.13greater than that of embryos derived from ADSCs-pCDsRed2-l and2.71greater than that of embryos derived from MDSCs-pCDsRed2-1(P<0.01).(3) Production and detection of transgenic cloned Arbas Cashmere goatsThe4-6pieces embryos of4-8cell stage were transplanted into ipsilateral fallopian tubes of receptors. Expression of the DsRed2gene in the fur of transgenic cloned Arbas Cashmere goats was detected using Western-blot analysis. The results showed that in this study, in ADSCs-pCDsRed2-1group, two transgenic cloned goats were survived. In MDSCs-pCDsRed2-1and FFCs-pCDsRed2-1groups, one goat was survived respectively. All of the transgenic cloned Arbas Cashmere goats expressed DsRed2.In summary, ADSCs, BMSCs and MDSCs lines of Arbas Cashmere goats were isolated, cultured and established in this study. Three kinds of adult stem cells in vitro were proliferative and still maintained good sternness and differentiation potential after multiple passages. And compared with the FFCs, transfection efficiency of these three types of adult stem cells were significantly higher than FFCs; while the random integration of exogenous genes did not affect their pluripotency. Finally, we had got four transgenic lambs came from different cell lines, and ADSCs, BMSCs and MDSCs did not significantly affect the transgenic cloning efficiency. |
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