转PRL基因小鼠遗传性状分析

doi:10.11843/j.issn.0366-6964.2016.09.015

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (9): 1861-1867.doi: 10.11843/j.issn.0366-6964.2016.09.015

转PRL基因小鼠遗传性状分析

任艳萍^1，2，谢亮亮¹，李海艳¹，石德顺^1* ，李湘萍^1*

（1.广西大学亚热带农业生物资源保护与利用国家重点实验室，南宁 530004； 2.遵义医学院基础医学院，遵义 563003）

收稿日期:2015-12-17 出版日期:2016-09-23 发布日期:2016-09-29
通讯作者: 石德顺，博士，研究员，主要从事胚胎工程研究，E-mail：ardsshi@gxu.edu.cn；李湘萍，博士，研究员，主要从事胚胎工程研究，E-mail：xiangpingli@163.com
作者简介:任艳萍(1986-)，女，河南人，博士，主要从事胚胎工程研究，E-mail：ypren@foxmail.com
基金资助:
国家转基因重大专项(2014ZX08007-001)；国家自然科学基金（31560632）；广西自然科学基金项目（2014GXNSFAA118084）

The Genetic Traits Analysis of PRL Transgenic Mice

REN Yan-ping^1，2，XIE Liang-liang¹，LI Hai-yan ¹，SHI De-shun^1* ，LI Xiang-ping ^1*

(1.State Key Laboratory of Subtropical Bioresource Conservation and Utilization，Guangxi University，Nanning 530004，China；2.School of Basic Medical Sciences，Zunyi Medical University，Zunyi 563003，China)

Received:2015-12-17 Online:2016-09-23 Published:2016-09-29

摘要/Abstract

摘要：

本研究以原核注射法生产的乳腺特异性表达PRL基因小鼠为模型，从外源基因遗传完整性、拷贝数和插入位点3方面对其进行遗传性状分析，为今后大型转基因动物的选育提供基础。采用基因组DNA PCR方法筛选发生外源基因片段部分缺失的小鼠，并进行Southern blot验证，以检测外源基因的遗传完整性；用绝对荧光定量PCR方法测定外源基因拷贝数，用Genome walking方法分析外源基因整合位点。结果显示，外源基因片段部分缺失率为1.91%；整合十几个拷贝外源基因的小鼠在便携UV灯下可见绿色荧光，整合2个拷贝的小鼠无绿色荧光；在NW_001073945.1序列的2 025~2 026 bp和NW_001030574.1序列的10 760 020~10 760 021 bp处插入的外源基因可正常表达。以上结果说明，外源PRL基因在原核注射法生产的转基因小鼠中可稳定遗传；在一定范围内，外源基因的表达量与拷贝数呈正相关；附近无其他基因的外源基因插入位点有利于外源基因的表达。

关键词: 转基因动物, 遗传性状, 原核注射, 外源基因插入位点, 拷贝数

Abstract:

In order to provide the foundation for breeding large transgenic animals in futher，in this study，the genetic traits of mammary gland-specific PRL transgenic mice produced by pronuclear microinjection，were analyzed from the genetic integrity，the copy number and the inserting site of exogenous gene.The transgenic mice deleted partially exogenous gene were screened by genomic DNA PCR method，and were verified by Southern blot to detect the genetic stability of exogenous gene.The copy number of exogenous gene in transgenic mice was detected by absolute real-time fluorescence quantitative PCR method.The inserting site of exogenous gene in transgenic mice was analyzed by Genome walking method.The results showed that the losing rate of exogenous gene fragment was 1.91%.The green fluorescence signal was observed under the long-wave UV lamp in the transgenic mice integrated higher copies of exogenous gene，while not observed in the transgenic mice integrated 2 copies of exogenous genes.The exogenous gene expressed normally in the 2 025-2 026 bp site of NW_001073945.1 gene and the 10 760 020-10 760 021 bp site of NW_001030574.1 gene.The result indicated that the exogenous gene could be inherited stablely in the transgenic mice produced by pronuclear microinjection，the expression level was positively correlated with the copy number of exogenous gene in certain range，and it was beneficial for the exogenous gene expression while there was no other genes nearby the inserting site.

Key words: transgenic animal, genetic traits, pronuclear microinjection, the inserting site of exogenous gene, copy number

中图分类号:

S865.13

任艳萍，谢亮亮，李海艳，石德顺，李湘萍. 转PRL基因小鼠遗传性状分析[J]. 畜牧兽医学报, 2016, 47(9): 1861-1867.

REN Yan-ping，XIE Liang-liang，LI Hai-yan，SHI De-shun，LI Xiang-ping. The Genetic Traits Analysis of PRL Transgenic Mice[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1861-1867.

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转PRL基因小鼠遗传性状分析

The Genetic Traits Analysis of PRL Transgenic Mice

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