Construction of an Efficient Delivery Vector Based on Fluorinated Polyethyleneimine for Transfection of Cdh23 Full-length Plasmid in HEI-OC1 Cell
10.13865/j.cnki.cjbmb.2025.06.1030
- VernacularTitle:基于氟化聚乙烯亚胺构建高效递送载体实现Cdh23全长质粒在HEI-OC1细胞中的转染
- Author:
Bing-Qian LI
1
;
Mu-Lan LI
;
Miao XIA
;
Zhen LIU
;
Lan WANG
;
Peng MA
Author Information
1. 滨州医学院特殊教育与康复学院听觉言语康复科学研究院,山东 烟台 264003
- Publication Type:Journal Article
- Keywords:
fluorinated-polyethyleneimine;
cadherin 23 gene;
large gene overexpression;
HEI-OC1 cell;
Usher syndrome type 1D
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(9):1349-1359
- CountryChina
- Language:Chinese
-
Abstract:
The CDH23 gene is a pathogenic mutant gene of the USH1D subtype in Usher syndrome.In this study,two wild-type Cdh23 full-length plasmids(～16 kb)with different promoters were construc-ted,and fluorinated polyethylene imine(FPEI)was used as a delivery vector to transfect the house ear institute-organ of corti 1(HEI-OC1)and the optimal expression plasmid was obtained by evaluating the transfection efficiency in vitro.Firstly,the results of the synthesis of FPEI were analyzed using Fourier transform infrared absorption spectroscopy to prove the successful synthesis of FPEI.After that,the plas-mid encapsulation ability of FPEI and the surface potential and hydration diameter of the formed comple-xes were characterized by agarose gel blocking assay,Zeta potential assay,and dynamic light scattering assay.It was found that FPEI had good plasmid encapsulation ability,and the FPEI plasmid complexes were all positively charged at high mass ratio,with the distribution of particle sizes in the range of 100-300 nm.The low cytotoxicity and high transfection efficiency of FPEI in HEI-OC1 cells were verified by Cell Counting Kit-8(CCK-8)and flow cytometry.Comparing FPEI with Lipofectamine 3000 and differ-ent quality PEI(25K,40K)transfection reagents,the transfection efficiency of FPEI was found to be significantly better than that of the traditional transfection reagents.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot results showed that the CAG promoter was better than the CMV promoter,which could be used as the optimal expression plasmid for the subsequent in vivo experi-ments.In addition,it was verified by cellular immunofluorescence that CDH23 was mainly distributed in the cytoplasm after overexpression.The above results demonstrated that FPEI can be used as an efficient delivery vector for in vitro overexpression of large genes represented by Cdh23,which provides an impor-tant experimental basis for subsequent in vivo gene therapy of USH1D syndrome.
