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姜自琴, 吴寒宇, 田菁, 李宁, 胡晓湘. 2020: 慢病毒靶向感染PGCs制备转基因鸡的方法研究. 动物学研究, 41(3): 281-291. DOI: 10.24272/j.issn.2095-8137.2020.032
引用本文: 姜自琴, 吴寒宇, 田菁, 李宁, 胡晓湘. 2020: 慢病毒靶向感染PGCs制备转基因鸡的方法研究. 动物学研究, 41(3): 281-291. DOI: 10.24272/j.issn.2095-8137.2020.032
Zi-Qin Jiang, Han-Yu Wu, Jing Tian, Ning Li, Xiao-Xiang Hu. 2020: Targeting lentiviral vectors to primordial germ cells (PGCs): An efficient strategy for generating transgenic chickens. Zoological Research, 41(3): 281-291. DOI: 10.24272/j.issn.2095-8137.2020.032
Citation: Zi-Qin Jiang, Han-Yu Wu, Jing Tian, Ning Li, Xiao-Xiang Hu. 2020: Targeting lentiviral vectors to primordial germ cells (PGCs): An efficient strategy for generating transgenic chickens. Zoological Research, 41(3): 281-291. DOI: 10.24272/j.issn.2095-8137.2020.032

慢病毒靶向感染PGCs制备转基因鸡的方法研究

Targeting lentiviral vectors to primordial germ cells (PGCs): An efficient strategy for generating transgenic chickens

  • 摘要: 慢病毒作为制备转基因鸡的经典工具, 通常利用水泡型口炎病毒包膜蛋白(Vesicular stomatitis virus G, VSVG) 进行病毒包装, 经包膜后的病毒具有广泛的细胞感染性。然而这种非特异感染细胞的性质导致利用慢病毒制备转基因鸡性腺嵌合率较低,进而引起转基因后代筛选效率偏低。为解决这一科学问题,本研究利用改装后的Sindbis病毒包膜蛋白 (M168) 包装慢病毒, 在特异抗体的介导下靶向感染鸡的原始生殖细胞 (Primordial germ cells, PGCs) 制备转基因鸡, 以期提高转基因鸡制备及筛选效率, 加速转基因禽类的研究进展。本研究首先建立了以第二代慢病毒包装系统为主体的病毒包装体系,并优化了靶向感染系统中抗体浓度与M168假型化慢病毒(M168-LVs)之间最佳的结合比例;随后通过体外分离培养PGCs,并对其进行病毒靶向感染研究。结果显示SSEA4抗体介导的M168-LVs与VSVG-LVs相比, 能靶向感染PGCs而不感染饲养层细胞和鸡成纤维细胞(DF-1);最后结合三期换壳法,利用SSEA4抗体介导的M168-LVs显微注射到鸡胚盘下腔进行转基因鸡制备研究。结果显示SSEA4抗体介导的M168-LVs转染的鸡胚中有50.0%–66.7%的性腺表达GFP, 转染效率与对照组VSVG-LVs相比提高了30.0%–36.7%,说明SSEA4抗体介导的M168-LVs在体内不仅能够靶向感染PGCs, 还提高了性腺的嵌合比率, 为进一步进行转基因鸡的制备研究提供了理论依据,并为加速转基因禽类的研究进展提供方法基础。

     

    Abstract: Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells (PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein (termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein (GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

     

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