ABSTRACT
Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions. Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase (Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids (PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon (PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography (GC). The expression level of Δ15 Des in mice was detected by quantitative PCR (qPCR) and Western blotting (WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes. This further verified the efficient transduction efficiency of the PiggyBac transposon system.
Subject(s)
Animals , Mice , Caenorhabditis elegans/genetics , Fatty Acid Desaturases/genetics , Fatty Acids , Fatty Acids, Omega-3 , Mice, TransgenicABSTRACT
@#Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.
ABSTRACT
OBJECTIVE To study the methodology of achieving stable co-expression of drug-metab?olizing enzymes in the HepG2 cells by the piggyBac (PB) transposon system. METHODS N-terminal attachment of enhanced green fluorscent protein plasmid (pEGFP- N2) and 2A peptide linked recombinant PB transposon plasmid containing dual-genes encoding drug metabolizing enzymes cyto?chrome P450 3A4 (CYP3A4) and CYP2C19 (pPB-CYP3A4-2A-2C19) were transfected into HepG2 cells respectively by Lipofectamine?LTX reagent, GenJetTM (Ver.Ⅱ) reagent and Neon?Transfection System reagent, which were widely used for large-sized DNA fragments transfection. 48 h later, the transfection efficiency and cell toxicity were detected and compared between the three methods so as to find a method with relatively high efficiency and low toxicity for later transfection.Then,three groups of recombinant PB transposons-single-gene transposon (PB-CYP3A4), 2A peptide linked dual-gene transposon (PB-CYP3A4-2A-2C19) and multiple single-gene transposon mixture〔PB-CYP3A4, PB-CYP2C8, PB-CYP2A6, organic anion transporting polypeptide 1B1 PB transposon (PB-OATP1B1)〕-were transfected into HepG2 cells respectively with the above established method.The puromycin (Puro)-resistant and GFP positive cell clones were picked up and further cultured. The mRNA, protein and metabolic levels of drug-metabolizing enzymes in monoclonal cell lines were detected by quantitative real-time PCR,Western blotting and high performance liquid chromatography-tandem mass spectrometry respectively after screening by Puro and green fluorescence. Comparisons of different groups were made using statistical analysis. RESULTS The comparison of three different transfection methods indi?cated that the transfection efficiency of GenJetTMwas up to(94.2±2.5)% and (89.3±3.3)%,significantly higher than those of the other two methods (P<0.01), along with lower cytotoxicity. Then GenJetTMwas chosen for later transfection. In the Puro-resistant monoclonal cell lines of single transposon PB-CYP3A4,PB-CYP3A4-2A-2C19 groups,the mRNA,protein and enzyme activity levels of drug-metabo?lizing enzymes were significantly increased respectively.The recombinant transposon (PB-CYP3A4-2A-2C19) containing 2A peptide could achieve stable and efficient co-expression of two metabolizing enzymes CYP3A4 and CYP2C19,while the expression of drug-metabolizing enzymes remained unbal?anced and random in those of multiple single-gene transposon mixture group (PB-CYP3A4, PB-CYP2C8,PB-CYP2A6,PB-OATP 1B1)(CYP3A4 was expressed in some cell clones only).CONCLUSION GenJetTM could be an effective method for the PB recombinant transposon transfection into HepG2 cells, by which the PB transposon could mediate stable expression of drug-metabolizing enzymes. In terms of multi-gene expression,a low and unbalanced expression is found by multiple transposons co-transfection method,which is different from that by virus mediated method.In contrast,mono-PB trans?poson linked by 2A peptide can achieve stable expression of multi-genes.
ABSTRACT
Objective@#An innovative technique was established to rapidly construct various cell lines that could be induced to express multiple influenza A virus (IAV) proteins.@*Method@#The HA protein genes of multiple IAVs were cloned into the Cumate-induced expression system which was positioned between two PiggyBac transposon sites. These HA plasmids were transfected into the HEK293A cell line with the PiggyBac transposase plasmids. The transfected cells were screened with puromycin, and after that the corresponding virus proteins were induced with Cumate.@*Results@#The results of flow cytometry and Western blotting showed that the virus proteins were expressed in most of the cells in corresponding lines after the induction of Cumate.@*Conclusion@#Cell lines which were inducible to express IVA HA protein can be efficiently constructed by using the PiggyBac transposon system.