磁小体介导制备转基因猪模型

doi:10.3969/j.issn.1674-5817.2015.04.005

摘要/Abstract

摘要： 目的以磁小体(BMP)和聚乙烯亚胺(PEI)为载体,通过睾丸注射精子介导法建立转基因猪模型。方法将实验室保存的脂蛋白磷脂酶A2(Lp-PLA2)绿色荧光蛋白(GFP)质粒与BMP和PEI 混匀,制备BMP-PEI- PLA2-GFP混合物,采用睾丸多点注射法接种巴马小型猪, 于接种后第2周、4周、6周至第20周采集精液,分别用RT-PCR和免疫荧光鉴定外源基因的表达情况; 取阳性精液,对母猪实施人工授精; PCR及RT-PCR对所产仔猪进行鉴定。结果精液中GFP基因于接种后第4周首现并持续至第16周; 荧光显微镜检查可观察到GFP蛋白主要表达于精子顶体后区及精子尾颈部; 经人工授精所产21头仔猪中,9头猪用PCR可检测GFP片段,用RT-PCR检测PLA2表达,该9头猪的表达量明显高于其它猪,总阳性率达42.86%。结论 BMP和PEI结合可作为良好的携带外源基因的非病毒性载体,用该载体通过精子介导法可以达到迅速制备转基因猪的目的, 其成本低、方法简单、便捷。

关键词: 猪, 转基因, 磁小体(BMP), 聚乙烯亚胺(PEI), 精子介导的基因转移

Abstract: Objective To establish sperm-mediated transgenic pigs models by intratestis injection with bacteria magnetic particles (BMP) and polyethylenimine (PEI) as a vector. Method The Lp-PLA2- GFP plasmid were mixed with BMP-PEI to BMP-PEI- PLA2-GFP, which were delivered into Bama Mini-pigs by intratestis injection. The sperm were collected in 2 weeks, 4 weeks, 6 weeks to 20 weeks after inoculation, and the GFP gene were detected by RT-PCR and immunofluorescence. The positive sperms will be transferred through artificial insemination to sows to establish transgenic pigs. The piglets were detected by RT-PCR to GFP gene and Real-time PCR to Lp-PLA2 gene. Results The GFP gene was first appearance in semen in 4 weeks after inoculation, and sustained through 16 weeks. The GFP protein mainly expressed in the sperm acrosome, sperm tail and neck area were observed by fluorescence microscopy. Through artificial insemination, 21 piglets were born, 9 of which were found with GFP segments, and the expression of PLA2 was obviously higher than that of other pigs, with a total positive rate of 42.86%. Conclusion The BMP combined with PEI can serve as a good non viral vector carrying exogenous gene, and sperm-mediated method with the vector can achieve the purpose of rapid preparation of transgenic pigs, its cost is low, the method is simple and convenient.

Key words: Pig, Transgenic, Bacteria magnetic particles (BMP), Polyethylenimine (PEI), Sperm-mediated gene transfer

中图分类号:

Q95-33

陈克研, 陈振文, 董婉维, 郑志红. 磁小体介导制备转基因猪模型[J]. 实验动物与比较医学, 2015, 35(4): 288-293.

CHEN Ke-yan, CHEN Zhen-wen, DONG Wan-wei, ZHENG Zhi-hong. Producing Transgenic Pig by Bacteria Magnetic Particles[J]. Laboratory Animal and Comparative Medicine, 2015, 35(4): 288-293.

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