Expression Of Genes Encoding Formyl-coa Transferase And Oxalyl-coa Decarboxylase From Chinese Oxalobacter Formigenes In Fetal Heptic Stem Cells With Transfection Of Recombinant Lentiviral Vectors

0BJECTIVE: Oxalobacter formigenes(Ox.F) is an obligate anaerobe which can degrades oxalates using oxalyl-CoA decarboxylase and formyl- CoA transferase encoded by the OXC and FRC genes, respectively.To clone FRC and OXC from Oxalobacter formigenes in Chinese people intestines, then construct and manufacture recombinant lentiviral expressing vectors pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED,providing a basis for further study on transgenic hepatic stem cells for treatment for hyperoxalurias.MATERIALS AND METHODS: The Oxalobacter formigenes were isolated from Chinese fecal specimens previously and stored in our laboratory. Then template DNA from Chinese Oxalobacter formigenes was isolated by bacterial genome extraction kit , the cDNA of FRC and OXC were cloned by PCR with Taq DNA polymerases and then ligated with pMD18T simple vectors after retrieve and purification. The ligation products were transformed into competent E.coli DH5α.The positive recombinant clones were selected and identified by a complementation, restriction endonuclease digestion. The cloning vector first digested with BamHⅠand the lentivirus vectors pLenti6.3/v5 DEST-IRES-EGFP and pLenti6.3/v5 DEST-IRES- DsRED were ligated and transformed respectivly. The DNA sequence analysis were performed to confirm the recombinant lentiviral vector and the titer of constucted lentivirus vectors were detected. RESULTS: Two fragments of 1287 bp and 1707bp was obtained by PCR respectivly. The enzyme and PCR analyses revealed that the correct FRC and OXC cDNA was cloned. The sequence of pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED was identical to the sequence of cloned cDNA respectivly.HEK293T cells were observed green and red fluorescent after 24h transfection, Two type of virus titers were 1.15×108TU/ml and 9.75×107TU/ml respectively.CONCLUSIONS: FRC and OXC was cloned correctly and the recombinant lentiviral vector pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED was constructed successfully, then high titer of Lentivirus was prepared OBJECTIVE: To isolate and identificate hepatic stem cells by mechanical separation and enzymatic digestion methods from rat fetal liver and observe green fluorescent protein (GFP) expression after tranfection with pEGFP-C1 plasmid .MATERIALS AND METHODS: Mechanical separation and enzymatic digestion method were used to isolate fetal hepatic stem cells from fetal Sprague Dawley rat liver tissues following pregnant for 13.5 days. Half-amount medium replacement was used for purify isolated fetal liver cells following cultured in specific medium. Immunofluorescence technique, Real-time PCR and Western Blot was used to identify adherent cells.The fetal hepatic stem cells of 3 passage were tranfected by electroporation with pEGFP-C1 plasmid .RESULTS: The isolated fetal hepatic stem cells adhered to the culture plastic and presented with round or oval shape after 24-hours cultivation in vitro. Isolated cells were almost equal circular after 2 days cultivation. Cells grew into a colony which was constructed by 3 or 5 cells at 3 days, with cell diameter of 6-10μm; cell colony became more bigger and constructed by 10 cells; the colony was composed of dense round cells of various sizes with clear boundary at 5 days. At 8 days, they grew like epithelium cell. At 12 days, cells became big and extended like fried egg with irregular forms, especially in cytoplasm. Following passage, there were no significant changes in cell amplification speed. Cells still presented epithelium-like shape at the passage 3. The adhered cells at 5 days following primary incubation were positively for human stem cell factor receptor CD34 and cytokeratin 19 using immunofluorescence technique and Western Blot. Real-time PCR detected the AFP and ALB expressed in the adhered cells. GFP can stably express in transgenic cells after pEGFP-C1 plasmid transfection.CONCLUSIONS: The mechanical separation and enzymatic digestion methods is a simple, economic and effective method to isolate hepatic stem cells from rat fetal liver. The transgenic fetal hepatic stem cell with GFP is the basis of hepatic stem cell transplantation for curation of PHs. OBJECTIVE: To investigate the oxalate-degrading effects of fetal hepatic stem cells with lentivirul vector transfection of genes encoding oxalate-degrading enzyme from Chinese Ox.F in vitro.MATERIALS AND METHODS: We applied the pre-constucted over-expression lentiviral vector pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED to transfect fetal hepatic stem cells, and then sort positive cells by flow Cytometry.Then detect the expression of oxalate-degrading enzyme genes used Real-time PCR and western Blot. Finally, The oxalate-degrading function of transgenic cells was analyzed by ion chromatography.RESULTS: The hepatic stem cells were successfully transfected with Lentiviral overexpression vector pLenti6.3-OXC-IRES-DsRED and pLenti6.3-FRC-IRES-EGFP. Real-time PCR and Western Blot respectively detected in the expression of FRC and the OXC in gene and protein levels. Compared with the three cell groups,The concentration of oxalate in culture medium of pLenti6.3-FRC-IRES-EGFP transgenic cells [(1.37±0.02)g/L] was lower than that of blank vector transgenic cells control group [(1.57±0.01)g/L]and blank control group [(1.58±0.03)g/L] (P < 0.0 1).CONCLUSIONS: Lentiviral vector transfection of oxalate-degrading enzyme genes is an effective way which make fetal heptic stem cells have potency of oxalate-degrading sustainably, It lay a good foundation for cell transplantation in the management of Hyperoxaluria.