Effect of GC-rich DNA fragment on the level of transgene expression in Chinese hamster ovary cells and its position effect
10.7683/xxyxyxb.2017.09.004
- VernacularTitle:富含GC的DNA片段对中国仓鼠卵巢细胞中转基因表达的影响及其位置效应
- Author:
Sijia CHEN
;
Chunpeng ZHAO
;
Junhe ZHANG
;
Xiaoyin WANG
;
Tianyun WANG
- Keywords:
GC-rich DNA fragment;
transgene;
position effect
- From:
Journal of Xinxiang Medical College
2017;34(9):794-797
- CountryChina
- Language:Chinese
-
Abstract:
Objective To analyze the effect of GC-rich DNA fragments on the level of transgenic expression in Chinese hamster ovary (CHO) celts and its position effect.Methods The synthetic DNA fragment with GC-rich was cloned into the 5'or 3'or both 5'and 3'ends of expression cassette of expression vector.Three new expression vectors (pIRES-G1,pIRES-G2 and pIRES-G3) which was inserted with the GC-rich DNA fragments in different position were transfected CHO ceils,respectively,and then was observed under fluorescence microscope;the control vector was pIRES-EGFP.Stable transfected cell lines were screened under G418,and enhanced green fluorescent protein(EGFP) expression was analyzed by flow cytometry and the transgenic copy number was detected by quantitative real-time quantitative polymerase chain reaction (qRT-PCR).Results Three expression vectors with a GC-rich DNA fragments in different position were constructed successfully.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector could obviously improve the expression level of vector in CHO cells;and the expression level of the stably transfected CHO cells increased 1.39 fold and 1.32 fold compared to the control vector,respectively;the transgene copy number increased 1.32 fold and 1.24 fold compared with the control vector.While the insertion of GC-rich DNA fragments at 5'end of expression cassette had no obvious effect on the level of gene expression.Conclusion The role of DNA fragment with GC-rich in improving the transgenic expression of CHO cells is related to its position in the vector.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector can improve transgenic expression.
