早期胚胎血管显微注射慢病毒载体制备转基因禽类的研究

doi:10.11843/j.issn.0366-6964.2015.12.009

摘要/Abstract

摘要：

根据禽类独特的生殖生理特点及特殊的胚胎发育模式，把原始生殖细胞法、显微注射法和慢病毒载体法3种转基因技术结合起来，以期探索出简便、高效的生产转基因禽类新方法。在鹌鹑胚胎发育至第13~15期，血液循环中的原始生殖细胞（Primordial germ cells，PGCs）数目达到最高值，血管显微注射携带增强型绿色荧光蛋白基因（Enhanced green fluorescent protein，eGFP）的人类免疫缺陷病毒I型（Human immunodeficiency virus-1，HIV-1）慢病毒载体，每枚胚胎注入1 μL，滴度为1×10⁹ TU•mL^-1。80枚鹌鹑种蛋，孵化出雏48只，孵化率为60%；对新生鹌鹑解剖后，在荧光显微镜下检测，其喙部、羽毛、眼部、大脑、血管、心、肝、脾、肺、肾、腺胃、肠系膜、小肠、大肠、输卵管、肌肉及爪上检测到绿色荧光蛋白表达，特别是在性腺中观察到绿色荧光蛋白广泛性表达。G0代28只雄性鹌鹑中，成功采集到21只精液，其中5只个体精子基因组经聚合酶链式反应（Polymerase chain reaction，PCR）检测阳性，阳性率为23.8%（5/21）；在5只阳性个体的G1代46只后代中，有6只经PCR及印迹杂交（Southern blot）检测均为阳性，阳性率为13.0%（6/46）。利用胚胎发育至第13~15期血管显微注射慢病毒载体能够有效感染此时期迁移的PGCs，获得性系嵌合体个体，最终成功获得转基因后代。该方法简便、高效的生产出转基因禽类，必将为禽类转基因研究提供一种新的思路和方法。

Abstract:

This study was aimed to identify the availability of early embryo blood vessels microinjection of lentiviral vector as a new，effective way in making transgenic birds，here，we combined some of the transgenic technology such as primordial germ cells(PGCs)，microinjection and lentiviral vector together according to the characteristic of bird development.The number of PGCs in the bloodstream was maximal when the chicken embryoes developed to Hamburger-Hamilton stage 13-15(HH13-15)，1 μL lentiviral vector，containing an enhanced-green fluorescent protein(eGFP)，was microinjected into the blood vessels of quail embryos at stage HH13-15 with a titer of 1×10⁹TU•mL^-1.A total of 80 embryos were injected and 48 quails(60%) were successfully hatched.In newly hatched quails，eGFP expression was shown as the presence of green fluorescence in the beak，feather，eye，brain，blood vessels，heart，liver，spleen，lung，kidney，glandular stomach，mesenterium，small intestine，large intestine，oviduct，muscle and claws，especially in gonads.In 5 out of 21 mature G0 male quails，the semen was eGFP-positive(5/21，23.8%)，as detected by polymerase chain reaction(PCR).Southern blot and genetic analyses revealed that in the 46 G1 offspring produced by G0 quail，6 were transgenic(6/46，13.0%) according to the PCR and Southern blot results.In conclusion，transgenic germ line chimeras was successfully generated by injection of lentiviral vector into embryonic blood vessel at stage HH 13-15.It indicated that infection of PGCs with lentiviral vector via direct injection into blood vessels has the potential to provide a more convenient and efficient way to produce transgenic birds.

中图分类号:

S814.8

张自富，赵瑜，刘锦妮，李洵，彭新亮，章平，胡静，贺东阳，常姣姣，谷梦迪，金楠楠，王亚芳. 早期胚胎血管显微注射慢病毒载体制备转基因禽类的研究[J]. 畜牧兽医学报, 2015, 46(12): 2185-2191.

ZHANG Zi-fu，ZHAO Yu，LIU Jin-ni，LI Xun，PENG Xin-liang，ZHANG Ping，HU Jing，HE Dong-yang，CHANG Jiao-jiao，GU Meng-di，JIN Nan-nan，WANG Ya-fang. Producing Transgenic Avian by Microinjection of Lentiviral Vector into the Early Embryo Blood Vessels[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2185-2191.

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参考文献

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