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| Effect of Different Treatments of Rabbit(Lepus brachyurus) Embryos on Isolation and Culture of ES-like Cells |
| To explore the effects of different treatment of rabbit(Lepus brachyurus)embryos on developmental potential of early rabbit embryos in vitro and select the better culture system fit for isolation and cultivation Rabbit embryonic stem(ES)-like cells and rabbit embryos were treated with three different kinds of treatments. Rabbit ES-like cells adherence, clone and growth behavior were observed, and the obtained rabbit ES-like cells were detected by AKP, RT-PCR detection of ES cells's transcription factors and differentiation ability in vitro. The results showed that the embryos which were treated with pricking zona pellucida, cutting and dispersing attached and proliferated better easily, comparing with the control group(53.8% vs 5.6%, 88.9% vs 5.6%, respectively)(P<0.05). Maybe, the zona pellucida and outer albumen layer was mainly obstacle for postponing the connection between embryos and mouse embryonic fibroblast (MEF) feeder. The blastulas attachment rate of cutting and dispersing embryos was significantly higher than that of pricking zona pellucida(88.9% vs 53.8%)(P<0.05), the former could effectively shorten the time of attachment, but ICM formation rate of the group of pricking zona pellucida was significantly higher than that of the group of cutting and dispersing embryos(48.8% vs 40.0%)(P<0.05). The clone forming efficiency of F1 and F2 of rabbit ES-like cells of the group of pricking zona pellucida was significantly higher than that of the other two groups(35.7% vs 24.8% vs 0%, 33.9% vs 15.2% vs 0%, respectively)(P<0.05), and the rabbit ES-like cells obtained were subcultured to 30 passages in vitro. ICM of pricking zona pellucida possessed highly development potential, which kept a complete ICM cluster. The obtained rabbit ES-like cells were examined, the results showed that rabbit ES-like cell colonies stained by alkaline phosphatase(AKP) were strongly positive. RNA of amplification pluripotent genes(GAPDH, Nanog and Sox2)of rabbit ES-like cell colonies were detected, the size of target fragments were consistent as expected by RT-PCR detection. Rabbit ES-like cells naturally differentiated into embryoid bodies(EBs) originated from three germ layers in vitro. It turned out that pricking zona pellucida would be better beneficial to improve the efficiency of embryonic stem cells isolated from rabbit embryos and suitable for keeping the rabbit ES-like cells culture and proliferation in vitro. |
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Abstract To explore the effects of different treatment of rabbit(Lepus brachyurus)embryos on developmental potential of early rabbit embryos in vitro and select the better culture system fit for isolation and cultivation Rabbit embryonic stem(ES)-like cells and rabbit embryos were treated with three different kinds of treatments. Rabbit ES-like cells adherence, clone and growth behavior were observed, and the obtained rabbit ES-like cells were detected by AKP, RT-PCR detection of ES cells's transcription factors and differentiation ability in vitro. The results showed that the embryos which were treated with pricking zona pellucida, cutting and dispersing attached and proliferated better easily, comparing with the control group(53.8% vs 5.6%, 88.9% vs 5.6%, respectively)(P<0.05). Maybe, the zona pellucida and outer albumen layer was mainly obstacle for postponing the connection between embryos and mouse embryonic fibroblast (MEF) feeder. The blastulas attachment rate of cutting and dispersing embryos was significantly higher than that of pricking zona pellucida(88.9% vs 53.8%)(P<0.05), the former could effectively shorten the time of attachment, but ICM formation rate of the group of pricking zona pellucida was significantly higher than that of the group of cutting and dispersing embryos(48.8% vs 40.0%)(P<0.05). The clone forming efficiency of F1 and F2 of rabbit ES-like cells of the group of pricking zona pellucida was significantly higher than that of the other two groups(35.7% vs 24.8% vs 0%, 33.9% vs 15.2% vs 0%, respectively)(P<0.05), and the rabbit ES-like cells obtained were subcultured to 30 passages in vitro. ICM of pricking zona pellucida possessed highly development potential, which kept a complete ICM cluster. The obtained rabbit ES-like cells were examined, the results showed that rabbit ES-like cell colonies stained by alkaline phosphatase(AKP) were strongly positive. RNA of amplification pluripotent genes(GAPDH, Nanog and Sox2)of rabbit ES-like cell colonies were detected, the size of target fragments were consistent as expected by RT-PCR detection. Rabbit ES-like cells naturally differentiated into embryoid bodies(EBs) originated from three germ layers in vitro. It turned out that pricking zona pellucida would be better beneficial to improve the efficiency of embryonic stem cells isolated from rabbit embryos and suitable for keeping the rabbit ES-like cells culture and proliferation in vitro.
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Received: 18 February 2014
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