Establishment and Preliminary Exploration of in vitro Pig-a Gene Mutation Assay Based on L5178Y Cells

doi:10.13560/j.cnki.biotech.bull.1985.2019-0855

Abstract

Abstract: The objective of this work isto establish and validatea method of detecting Pig-a gene mutation in mammalian cells based on mouse lymphoma cells L5178Y tk^+/--3.7.2C with negative compounds Glc,NaCl and positive compounds EMS,ENU,4-NQO,and B（a）P. Cytotoxicity evaluation was performed by calculating the relative doubling rate of the cells,and the antibody-labeled mutant cells were used to determine the flow detection template. The frequency of Pig-a gene mutation was detected on the 4th,8th,12th,16th and 20th day after EMS treatment,and the time point of the maximum mutation frequency was determined. The location of CD90 protein in the cells was detected by immunofluorescence technique,and the PCR was used for mutation site analysis. Results are as following：1）The RPD of the concentration groups set by Glc,NaCl,EMS,ENU,B（a）P,and 4-NQO were all > 50%. 2）The frequency of mutation of the Pig-a gene peaked on the 8th day after administration. The mutation frequency of Glc and NaCl in Pig-a gene was < 200×10^-6. There was no significant difference between each concentration group and solvent control group（P>0.05）. EMS,ENU,B（a）P,and 4-NQO resulted intheincrease in the mutationfrequency of the Pig-agene,and there was a significant difference compared with the solvent control group. 3）Immunofluorescence imaging showed that there was no CD90 protein on the surface of mutant cells,and wild type cells normally expressed CD45 and CD90 proteins. 4）Detection of gene mutation sites revealed the presence of 3mutation types：G→C,A→C,and C→T. In conclusion,this study successfully establishes a method of detecting the genotoxicity of in vitro Pig-a gene mutation in mouse lymphoma L5178Y cells with/without of S₉metabolic activation,providing new approach in vitro genotoxicity evaluation or screening genotoxicity in the early development stage of drugs.

Key words: Pig-a gene, L5178Y cells, in vitro gene mutation, flow cytometry, immunofluorescence, genotoxicity

WANG Ya-nan, WEN Hai-ruo, WANG Xue. Establishment and Preliminary Exploration of in vitro Pig-a Gene Mutation Assay Based on L5178Y Cells[J]. Biotechnology Bulletin, 2020, 36(1): 220-228.

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