CRISPR/Cas9介导RB1基因敲除及其在鸡前脂肪细胞分化、增殖中的功能研究

doi:10.11843/j.issn.0366-6964.2016.09.005

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (9): 1775-1784.doi: 10.11843/j.issn.0366-6964.2016.09.005

CRISPR/Cas9介导RB1基因敲除及其在鸡前脂肪细胞分化、增殖中的功能研究

张琦^1，2，黄娇娇³，杨彩侠¹，王宇祥^1，2，张慧^1，2，赵建国³，李辉^1，2*

（1.东北农业大学动物科学技术学院，哈尔滨 150030； 2.农业部鸡遗传育种重点实验室，哈尔滨150030；3.中国科学院动物研究所干细胞与生殖生物学国家重点实验室，北京 100101）

收稿日期:2016-03-22 出版日期:2016-09-23 发布日期:2016-09-23
通讯作者: 李辉，教授，E-mail：lihui@neau.edu.cn
作者简介:张琦（1990-），女，黑龙江齐齐哈尔人，硕士生，主要从事家禽遗传育种研究，E-mail：zhangxiaoqi0405@163.com
基金资助:
国家自然科学基金（青年科学基金）（31301960）；国家863项目(2013AA102501)；现代农业产业技术体系建设专项（CARS-42）

CRISPR/Cas9 Mediated RB1 Knockout and Its Impact on Chicken Preadipocytes Differentiation and Proliferation

ZHANG Qi ^1，2，HUANG Jiao-jiao ³，YANG Cai-xia¹，WANG Yu-xiang^1，2，ZHANG Hui ^1，2，ZHAO Jian-guo ³，LI Hui ^1，2*

（1.College of Animal Science and Technology，Northeast Agricultural University，Harbin 150030，China；2.Key Laboratory of Chicken Genetics and Breeding，Ministry of Agriculture，Harbin 150030，China；3.State Key Laboratory of Stem Cell and Reproductive Biology，Institute of Zoology，Chinese Academy of Sciences，Beijing 100101，China）

Received:2016-03-22 Online:2016-09-23 Published:2016-09-23

摘要/Abstract

摘要：

旨在研究RB1基因在鸡前脂肪细胞分化和增殖过程中的功能，本研究利用CRISPR/Cas9系统在鸡前脂肪细胞中敲除RB1基因，检测鸡前脂肪细胞的分化和增殖能力及相关标志基因的表达水平。结果表明，CRISPR/Cas9系统能够有效介导RB1基因的敲除并引起RB1基因翻译的提前终止，敲除效率可以达到10%。油红O提取比色和CCK-8的结果显示，敲除RB1基因后，鸡前脂肪细胞的分化能力减弱、增殖能力增强；qRT-PCR结果显示，RB1基因敲除之后脂肪细胞分化标志基因G0S2、FAS、A-FABP的mRNA表达水平下降，尤其是G0S2基因的表达水平在前脂肪细胞分化的各个阶段都表现为显著的下降。同时RB1基因敲除还引起细胞周期相关基因CyclinD1、E2F1、Ki67、PCNA的mRNA表达水平升高。初步推断，RB1基因可能是调控鸡前脂肪细胞分化和增殖的关键调节因子。

Abstract:

In order to study the role of RB1 gene on chicken preadipocyte differentiation and proliferation.CRISPR/Cas9 system was established and conducted to knockout the RB1 gene in the chicken preadipocyte.The proliferation and differentiation of chicken preadipocytes and the expression of related marker genes were detected.Results showed that the CRISPR/Cas9 system could effectively mediate RB1 knockout and cause truncated translation in chicken at the rate of 10%.Oil Red O and CCK-8 were used to evaluate the ability of differentiation and proliferation in chicken preadipocyte respectively.Knockout of RB1 in chicken preadipocyte resulted in enhanced proliferation capability and decreased differentiation capability in chicken preadipocyte.RB1 knockout decreased the transcriptional expression of adipocyte differentiation associated genes such as G0S2，FAS and A-FABP.Especially，the expression of G0S2 was significantly decreased at all stages of preadipocyte differentiation.Further， RB1 knockout increased the expression of cell proliferation associated genes such as CyclinD1，E2F1，Ki67 and PCNA, which was corresponded to the enhanced proliferation capability in the RB1 knockout cells.In summary，RB1 may be a key regulator on differentiation and proliferation of chicken preadipocyte.

中图分类号:

S831.2

张琦，黄娇娇，杨彩侠，王宇祥，张慧，赵建国，李辉. CRISPR/Cas9介导RB1基因敲除及其在鸡前脂肪细胞分化、增殖中的功能研究[J]. 畜牧兽医学报, 2016, 47(9): 1775-1784.

ZHANG Qi，HUANG Jiao-jiao，YANG Cai-xia，WANG Yu-xiang，ZHANG Hui，ZHAO Jian-guo，LI Hui. CRISPR/Cas9 Mediated RB1 Knockout and Its Impact on Chicken Preadipocytes Differentiation and Proliferation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1775-1784.

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CRISPR/Cas9介导RB1基因敲除及其在鸡前脂肪细胞分化、增殖中的功能研究

CRISPR/Cas9 Mediated RB1 Knockout and Its Impact on Chicken Preadipocytes Differentiation and Proliferation

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