Enhanced transgenesis by intracytoplasmic injection of envelope-free lentivirus
Enhanced transgenesis by intracytoplasmic injection of envelope-free lentivirus
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DOI：
10.1002/dvg.20289
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发表时间：
2007-04-01
期刊：
影响因子：
1.5
通讯作者：
Chan, Anthony Wing Sang
中科院分区：
文献类型：
--
作者：
Yang, Shang-Hsun;Agca, Yuksel;Chan, Anthony Wing Sang
我们通过胞浆内注射无包膜慢病毒证明了小鼠转基因的增强。在胚胎移植前，将携带泛素启动子 (LVU-GFP) 控制下的绿色荧光蛋白 (GFP) 基因的无包膜慢病毒显微注射到小鼠受精卵的细胞质中。通过 PCR 和 Southern 印迹分析证实 97% (31/32) 的成年小鼠是转基因的；在提取基因组 DNA 之前，通过荧光显微镜检查尾部剪断，所有创始小鼠均表达 GFP。 Southern 印迹分析揭示了从 1 到 32 的转基因插入数量。 F1 后代中转基因的存在证实了种系传播。正如预期的那样，当将无包膜的 LVU-GFP 显微注射到卵周间隙 (PVS) 时，会产生较低的转基因率 (2.2%；1/46)，因为细胞识别以及病毒包膜和靶细胞之间的膜融合是包膜病毒成功感染的先决条件。在这里，我们通过将无包膜慢病毒胞浆内病毒注射（INVI）到小鼠受精卵中，证明了无包膜慢病毒通过小鼠种系转基因高效建立稳定基因整合的能力。
We demonstrate enhanced transgenesis in mice by intracytoplasmic injection of envelope-free lentivirus. Envelope-free lentivirus carrying the green fluorescent protein (GFP) gene under the control of the ubiquitin promoter (LVU-GFP) was microinjected into the cytoplasm of mouse zygotes prior to embryo transfer. Ninety-seven percent (31/32) of the adult mice were confirmed transgenic by PCR and Southern blot analysis; all founder mice express GFP when tail snips were examined by fluorescent microscopy prior to genomic DNA extraction. Transgene insertion numbers ranging from I to 32 were revealed by Southern blot analysis. Germline transmission was confirmed by the presence of transgene in F1 offspring. As expected, a lower transgenic rate (2.2%; 1/46) resulted when envelope-free LVU-GFP was microinjected into the perivitelline space (PVS) because cell recognition followed by membrane fusion between the viral envelope and the target cell is prerequisite for successful infection by envelope viruses. Here we demonstrate the competence of envelope-free lentivirus in establishing stable gene integration by germline transgenesis in mice at high efficiency, by intracytoplasmic viral injection (INVI) of envelope-free lentivirus into mouse zygotes.