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Published on: April 7, 2023
[Construction of oxalate-degrading intestinal stem cell population in mice]
Zhang-Qun Ye1, Guan-Lin Liu, Zhi-Qiang Chen
1Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Objective:
The oxalate-degradation genes of Oxalobacter formigenes (Ox.F)-frc gene and oxc gene-were cloned and transfected into intestinal stem cell population of the mouse to make the latter obtain oxalate-degradation function.
Methods:
(1) The dicistronic eukaryotic expression vector, which could express oxc gene and frc gene in the same time, pIRES-oxc-frc was constructed. (2) The intestinal stem cell population of the mouse were isolated and cultured, and the function of the cell growth and differentiation was identified. (3) The cells were transfected with pIRES-oxc-frc. After selection by G418, the function of the cell growth and differentiation and the the expression of the objective genes were identified. (4) The concentration of the oxalate in the culture medium which was used to culture the transgenic cells was determined by ion chromatography to explore the oxalate-degradation function of the cells.
Results:
Ox.F could be isolated and cultured from the feces of Chinese people. Compared with the foreign reports, a certain morphologic variation of the Ox.F existed. But the oxc gene and frc gene showed high homology with the sequence reported in GenBank. The recombinant plasmid containing oxc gene and frc gene could successfully be transfected into the intestinal stem cell population of the mouse. The expression of the objective genes was normal. The concentration of the oxalate in the culture fluid of the transgenic intestinal stem cell population [(2.48 +/- 0.03 g/L)] was lower than those of the normal group [(2.69 +/- 0.01) g/L] and the control group [(2.69 +/- 0.01) g/L, P < 0.01].
Conclusion:
The oxc gene and the frc gene could be transfected into the intestinal stem cell population of the mouse, and the cells could be given oxalate-degrading function. The gene of prokaryocyte could be introduced into the eukaryocyte for a successful expression.
