Expression Of HEPO In Renal Tubular Cells Utilizing Uromodulin Promoter And 3' GPI Anchor Gene: Kidney-Based Transgenic Bioreactor

Objective:To generate a mammalian expression vector encoding the human erythropoietin cDNA, and expressed in Hela cells. Methods:cDNA encoding human erythropoietin was amplified using RT-PCR from total RNA extracted from human kidneys. Erythropoietin cDNA was cloned and completely sequenced to detect potential point mutations that were inadvertently introduced during RT-PCR. Site-directed mutagenesis was used to correct the three PCR mistakes.The recombinant vector was transfected into Hela cells to observe its expression by Western blot.Results:Although human erythropoietin cDNA was amplified and cloned, the clone contained three point mutations, all resulting in codon conversions. However, site-directed mutagenesis with correct, site-specific primers successfully corrected the mistakes, leading to a full-length, authentic clone.And then pcDNA3.1-EPO was successfully expressed in Hela cells proved by Western blot.Conclusions:A mammalian expression vector containing an authentic human erythropoietin cDNA has been successfully generated and expressed in Hela cells. Objective To investigate tissue-specific and transcriptional activity of Uromodulin gene promoter in cell lines.Methods EGFP was used as reporter to construct the expression vector pEGFP-URO and transfected into cell lines of HK-2,ACHN,T24 and HBZY-1,positive cells were observed as a green fluorescent under excitation light.EGFP activity of cells was detected by fluorescence microscopy and flow cytometry (FCM), compared to the vector pEGFP-N3. Results The activity of EGFP in HK-2 and ACHN cell lines was higher than that in the other cell lines (5～10/HP versus 0～1/HP),with 13.2% and 11.4% positive cells in HK-2 and ACHN cells, but no positive cell was found in the other cell lines.The vector pEGFP-N3 was high EGFP expression in all cell lines,with 23.08%(HK-2) and 19.24%(ACHN) positive cells. Conclusion Mouse URO gene promoter can be expressed in a tissue-specific way in human HK-2 and ACHN cells.The URO promoter transcriptional capicity is half of CMV promoter. Key words Uromodulin,Promoter,EGFP,Tissue-SpecificObjective Cloning the N-terminal signal peptide(SP) and the C-terminal GPI-anchor sequences of uromodulin,studying its membrane targeting function in HK-2 cells using EGFP as reporter gene.Methods Uromodulin full-length cDNA was amplified using RT-PCR from total RNA extracted from mice kidneys,then the N-terminal SP and the C-terminal GPI-anchor sequences of uromodulin were amplified using nest PCR.EGFP was used as reporter to construct the expression vector pN3 SP-EGFP-GPI and transfected into HK-2 cells,positive cells were observed as green fluorescent by fluorescence microscopy.Results The authentic C-terminal GPI-anchor sequences of uromodulin has been successfully generated.HK-2 cells were transfected by the fusion gene vector pN3 SP-EGFP-GPI and green fluorescent were detected by fluorescence microscopy,most of the green fluorescent was observed in the cell membrane. Conclusion The SP/GPI sequence can target heterologous protain into epithelial cell membrane and can be used in pharmaceutical proteins production.