生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (3): 31-.doi: 10.3969/j.issn.2095-1736.2020.03.031

• 研究报告 • 上一篇    下一篇

鸡bmp4过表达和敲除载体构建及活性验证

  

  1. 1. 扬州大学 江苏省动物遗传繁育与分子设计重点实验室, 扬州 225009；2. 扬州市职业大学 生物与化工工程学院, 扬州 225009
  • 出版日期:2020-06-18 发布日期:2020-06-10
  • 通讯作者: 左其生，博士，讲师，主要研究方向为动物胚胎工程与遗传工程研究，E-mail: 006664@yzu.edu.cn
  • 基金资助:
    国家自然科学基金（31872341）；国家重点研发计划（2017YFE0108000）；江苏省研究生科研与实践创新计划（KYCX19_2114）

Constructing and activity verifying of chicken bmp4 overexpression and knockout vectors

  1. 1. Key Laboratory of Animal Genetics and Molecular Design of Jiangsu Province, Yangzhou University,Yangzhou 225009; 2. School of Biological and Chemical Engineering,Yangzhou Polytechnic College, Yangzhou 225009, China
  • Online:2020-06-18 Published:2020-06-10
  • About author:李婷婷，硕士研究生，主要研究方向为动物遗传育种与繁殖，E-mail:3538550080@qq.com

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摘要： 在哺乳动物中，bmp4可以通过BMP信号通路调节原始生殖细胞(Primordial germ cells, PGCs）的形成，但其在鸡PGCs形成过程中的功能和机制仍不清晰。构建鸡bmp4的过表达及敲除载体，并转染DF-1细胞检测活性。通过同源重组技术构建pCDH-CMV-bmp4-EF1-copGFP重组载体，转染DF-1细胞，RT-qPCR检测bmp4的表达；根据bmp4 CDS序列设计3个敲除靶位点（gRNA），插入PGMLV-GM1构建gRNA序列的重组载体bmp4-sgRNA1、bmp4-sgRNA2和bmp4-sgRNA3。Cas9载体与bmp4-sgRNA载体共转染DF-1细胞，T7E1酶切和TA克隆试验检测敲除载体活性及敲除效率。扩增获得bmp4 CDS全长1546 bp，并成功构建pCDH-CMV-bmp4-EF1-copGFP载体，将其转染DF-1细胞后有绿色荧光表达（25.17%±0.007），RT-qPCR检测结果表明，与对照组相比转染pCDH-CMV-bmp4-EF1-copGFP的细胞中bmp4显著上调（P＜0.01）。3个gRNA重组载体构建成功，转染DF-1细胞后T7E1酶切检测均有敲除活性，TA克隆结果显示敲除效率分别为6.25%、12.5%和18.75%。结果说明鸡bmp4过表达及敲除载体构建成功，可为后期bmp4功能验证及机制解析等研究提供技术支持。

关键词: 鸡, bmp4, 过表达载体, 敲除载体

Abstract: Bmp4 regulates the formation of primordial germ cells (PGCs) through BMP signaling pathway in mammals, but its function and mechanism in the formation of chicken PGCs is still unclear. In this study, overexpression and knockout vectors of chicken bmp4 were constructed and transfected into DF-1 cells. The recombinant vector pCDH-CMV-bmp4-EF1-copGFP was constructed by homologous recombination technique, transfected into DF-1 cells, and the expression of bmp4 was detected by RT-qPCR. Three knockout target sites (gRNAs) were designed based on the bmp4 CDS sequence, and PGMLV-GM1 was inserted to construct the recombinant vectors bmp4-sgRNA1, bmp4-sgRNA2 and bmp4-sgRNA3. Cas9 vector was co-transfected into DF-1 cells with bmp4-sgRNA vector, and knockout vector activity and knockout efficiency were detected by T7E1 digestion and TA cloning assay. The full length of bmp4 CDS was 1546 bp, and the pCDH-CMV-bmp4-EF1-copGFP vector was successfully constructed. After transfected into DF-1 cells, it showed green fluorescence expression (25.17%±0.007). The results of RT-qPCR showed that bmp4 was significantly up-regulated in DF-1, after being transfected with pCDH-CMV-bmp4-EF1-copGFP compared with the control group (P<0.01). Three gRNA vectors were successfully constructed. The knockdown activity was detected by T7E1 digestion after transfection of DF-1 cells. The results of TA cloning showed that the knockout efficiencies were 6.25%, 12.5% and 18.75%, respectively. The results indicated that chicken bmp4 overexpression and knockout vector were constructed successfully, which could support the research of bmp4 function verification and mechanism analysis.

Key words: chicken, bmp4, overexpression vector, knockout vector

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