Construction of pEGFP-N1-TGF-β1 recombinant plasmid and transfected it into primary cultured AEC-Ⅱ of neonatal piglet using lipofectamine 2000
10.3760/cma.j.issn.1671-0282.2016.01.012
- VernacularTitle:建立pEGFP-N1-TGF-β1重组质粒和脂质体介导转染原代培养新生猪AEC-Ⅱ方法
- Author:
Yan XU
;
Bo SUN
- Publication Type:Journal Article
- Keywords:
Alveolar epithelium type Ⅱ cell;
Primary culture;
pEGFP-N1-TGF-β1 recombinant plasmid;
Transfection
- From:Chinese Journal of Emergency Medicine
2016;25(1):50-56
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct pEGFP-N1-TGF-β1 recombinant plasmid and transfect it into primary cultured neonatal piglet type Ⅱ alveolar epithelium cell (AEC-Ⅱ) by using lipofectamine 2000,in order to provide basis of methodology for producing recombinant plasmids for transplantation of transfected AEC-Ⅱ into ALI/ARDS animal model lungs.Methods PCR primers were designed to amplify the human TGF-β1 cDNA fragment from plasmid.XhoI and EcoRI were used for double digesting the empty plasmid pEGFP-N1 and cDNA fragment of human TGF-β1.Then the products of double enzyme digestion by using T4 DNA ligase were connected and transformed into DH5α and cultured over night for 16 hours.The structure of recombinant plasmid was identified by using PCR and base sequencing to verify the correctness of pEGFP-N1-TGF-β1 recombinant plasmid.It was then transfected into primarily cultured AEC-Ⅱ by lipofectamine2000 mediated transfection and cultured for another 48 hour.Plasmid DNA (pEGFP-N1-TGF-β1 recombinant plasmid) and lipofectamine 2000 were added into serum-free DMEM respectively,then DNA suspension and Lipofectamine 2000 suspension were blended together and added into cells.After 24-48 hours later,the expression level of enhanced green fluorescent protein (EGFP) was evaluated under fluorescence microscope.Results The structure of vector was verified as pEGFP-N1-TGF-β1 recombinant plasmid by using PCR and base sequencing.Green fluorescence found in some cells showed that the pEGFP-N1-TGF-β1 recombinant plasmids had been successfully transfected into primary cultured AEC-Ⅱ,however,the transfection efficiency still need tobe further improved such as repeating the transfection procedure once again or using adenovirus mediated transfection method to improve the efficiceny and to transplant the cells into animal lungs eventually.Conclusions pEGFP-N1-TGF-β1 recombinant plasmid was successfully constructed and,for the first time,transfected into primarily cultured AEC-Ⅱ of newborn piglets.This established method should be useful for investigation of therapeutic effect and outcomes of lung with experimental acute lung injury.
