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首页> 外文期刊>Korean Journal of Poultry Science >Production of hTPO Transgenic Chickens using Tetracycline-Inducible Expression System
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Production of hTPO Transgenic Chickens using Tetracycline-Inducible Expression System

机译：使用四环素诱导表达系统生产hTPO转基因鸡

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It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions (rtTA2sM2) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO)from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. Thebiological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenicchicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative tdtstablish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.
机译：众所周知，外源基因的过度表达可能在转基因动物中产生有害的生理或毒性作用。为了解决这些问题，我们构建了逆转录病毒载体，该载体设计为在四环素诱导型启动子的控制下表达外源基因。然而，由于传统的基于Tet的系统中的固有缺陷，四环素诱导表达系统（Tet系统）中的基因表达没有被完全调节，而是有点泄漏。当rtTA的高级版本（rtTA2sM2）和响应组件中的最小启动子（pTRE-tight）组合使用时，可以实现更严格可控的调节系统。在这项研究中，我们尝试使用逆转录病毒载体与Tet系统结合，从各种靶细胞和转基因鸡中生产人血小板生成素（hTPO）。 hTPO是血小板生成的主要调节剂，在造血干细胞的存活和扩增中具有重要作用。在体外的初步实验中，在鸡胚成纤维细胞（CEF）细胞中观察到了更高的hTPO表达和更严格的表达控制。我们还使用增殖依赖于hTPO的Mo7e细胞测量了hTPO的生物学活性。来自CEF的重组hTPO的生物学活性高于其商业对应物和来自其他靶细胞的hTPO。将重组逆转录病毒注射到未孵育的鸡胚的胚盘下方（X阶段）。在孵化21天后，从注入的138个卵中孵出15只小鸡。其中，有8只孵化小鸡的hTPO阳性。当向Go转基因鸡饲喂强力霉素（每1克饲料0.5 mg），四环素衍生物时，转基因鸡血的hTPO浓度为200 ng / mL。转基因雄鸡的精子中证实了转基因的种系传递。这些结果为大量生产具有商业价值和生物学活性的人细胞因子蛋白的生物反应器提供了有益的参考。

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