应用TALEN技术对兔CCR5基因进行靶向修饰

doi:10.3724/SP.J.1005.2014.0360

遗传 ›› 2014, Vol. 36 ›› Issue (4): 360-368.doi: 10.3724/SP.J.1005.2014.0360

应用TALEN技术对兔CCR5基因进行靶向修饰

唐成程¹, 张全军², 李小平¹, 樊娜娜², 杨翌¹, 全龙泉¹, 赖良学^1,2

1. 吉林大学动物科学学院, 长春 130062;
2. 中国科学院广州生物医药与健康研究院, 广州 510530

收稿日期:2014-02-18 修回日期:2014-03-18 出版日期:2014-04-20 发布日期:2014-03-20
通讯作者: 赖良学，博士，教授，研究方向：转基因与动物克隆。E-mail:lai_liangxue@gibh.ac.cn E-mail:lai_liangxue@gibh.ac.cn
作者简介:唐成程，硕士，专业方向：生物化学与分子生物学。E-mail:chengcheng.study@163.com
基金资助:
国家重大科学研究计划项目（编号：2011CB944203）和国家科技重大专项(编号：2009ZX10004-405)资助

Targeted modification of CCR5 gene in rabbits by TALEN

Chengcheng Tang¹, Quanjun Zhang², Xiaoping Li¹, Nana Fan², Yi Yang¹, Longquan Quan¹, Liangxue Lai^{1, 2}

1. College of Animal Science, Jilin University, Changchun 130062, China;
2. Guangzhou Institutes of Biomedicine and Health, Chinese Academyof Sciences, Guangzhou 510530, China

Received:2014-02-18 Revised:2014-03-18 Online:2014-04-20 Published:2014-03-20

摘要/Abstract

摘要：

缺少合适的能够被人免疫缺陷病毒1型(Human immunodeficiency virus 1, HIV-1)感染的动物模型是获得性免疫缺陷综合征/艾滋病(Acquired immunedeficiency syndrome, AIDS)疫苗和药物研发的瓶颈。HIV-1能在野生型兔子中形成持续感染, 在共表达人CD4和CCR5的兔细胞系中, HIV-1能高效复制, 并形成合胞体。若在家兔中高表达人CD4和CCR5, 就可能获得研究AIDS的理想动物模型。文章采用高效基因打靶技术—类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease, TALEN), 探讨在家兔CCR5基因位点定点敲入人CD4和CCR5, 获得能够感染HIV-1家兔模型的可能性。针对家兔CCR5基因, 设计了两对TALENs和一个同源打靶载体, TALEN mRNAs和DNA同源片段显微注射到家兔受精卵中, 体外培养3~5 d后, 收集24枚胚胎, 对胚胎的基因突变情况进行PCR和测序分析。结果显示, 在家兔CCR5位点, 24枚胚胎均发生了基因敲除, 5枚胚胎还发生了人CD4和CCR5基因敲入。该结果为建立艾滋病研究新动物模型奠定了基础。

关键词: 类转录激活因子效应物核酸酶(TALEN), 家兔, CCR5基因, 基因修饰

Abstract:

The lack of suitable animal model for HIV-1 infection has become a bottleneck for the development of AIDS vaccines and drugs. Wild-type rabbits can be infected by HIV-1 persistently and HIV-1 can be efficiently replicated resulting in syncytia in rabbit cell line co-expressing human CD4 and CCR5.Therefore, a rabbit highly expressing human CD4 and CCR5 may be an ideal animal model for AIDS disease study. In the present report, by using the efficient gene targeting technology, transcription activator-like effector nuclease (TALEN), we explored the feasibility of generating a HIV-1 model by knocking in human CD4 and CCR5 into rabbit genome. First we constructed two TALEN vectors targeting rabbit CCR5 gene and a vector with homologous arms. TALEN mRNAs and donor DNA were then co-injected into fertilized oocytes. After 3?5 days, 24 embryos were collected and used to conduct mutation analysis with PCR and sequencing. All the 24 embryos were detected with CCR5 knockouts and 5 were human CD4 and CCR5 knockins. Our results laid a foundation for establishing a new animal model for the study of AIDS.

Key words: transcription activator-like effector nuclease (TALEN), rabbit, CCR5, gene modification

唐成程, 张全军, 李小平, 樊娜娜, 杨翌, 全龙泉, 赖良学. 应用TALEN技术对兔CCR5基因进行靶向修饰[J]. 遗传, 2014, 36(4): 360-368.

Chengcheng Tang, Quanjun Zhang, Xiaoping Li, Nana Fan, Yi Yang, Longquan Quan, Liangxue Lai. Targeted modification of CCR5 gene in rabbits by TALEN[J]. HEREDITAS, 2014, 36(4): 360-368.

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应用TALEN技术对兔CCR5基因进行靶向修饰

Targeted modification of CCR5 gene in rabbits by TALEN

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