Site-Directed Mutagenesis Of TMEN43Gene And Function Analysis

Objectives:To introduce a point mutation in TMEM43gene and construct theeukaryotic expression vector, including wild type and mutant, then totransfect the eukaryotic cells with constructed wild type and mutantTMEM43gene tagged with GFP，and to observe the transfectionefficiency and identify the function affection, and to investigate the cellgrowth and apoptosis of transfected cells.Methods:The eukaryotic expression plasmid vector was constructed by DNArecombination techniques including cloning PCR, enzyme digestionand ligation. Eukaryotic expression vector inserted mutatedTMEM43C>T (p-mutTMEM43-EGFP-N1) was carried out by assay ofsite-directed mutagenesis kit based on plasmid (pTMEM43-EGFP-N1)which contains wild type TMEM43gene. The point mutation wasidentified with restriction enzyme digestion and sequencing. The pointmutation-introduced plasmid was applied to transform competentbacterial cells(DH-5α strain of E. coli,), and screened by selection plateresistance to kanamycin. The grown positive clones were picked upand propagated in LB broth for18-24hours. The propagated bacterial cells were used to preparation of plasmid genomic DNA by mini-prepkit. The extracted DNA was identified by restriction enzyme digestion,and then agarose electrophoresis and gel image print. The correctrecombined DNA was sequenced with sequencing cycle reactionfollowed by product purification and sequencer analyzer reading. TheDNA recombined plasmids containing wild type or mutant TMEM43gene were prepared by maxi-prep kit to get endotoxin-free eukaryoticexpression plasmids. The plasmids were sequenced in whole length ofgenomic DNA to ensure the recombined DNA sequence be correct. Thevectors containing TMEM43gene were applied to transfect HL-1cellswhich were cultured in Claycomb medium supplemented with10%fetal bovine serum, with Lipofectamine2000transfection reagent.Under fluorescent microscope, transfection efficiency was observed.With protein immunoblot, the wild type and mutant TMEM43expression in transfected cells was determined. MTT assay and flowcytometry were applied to detect cell growth and apoptosisrespectively.Results:1. Constructed eukaryotic expression vector pTMEM43-EGFP-N1wasidentified to be correct by means of single digestion of Age I or Xho I,or double enzyme digestion of Age I and Xho I. The linear fragmentsizes digested DNA were4.3kb and1.3kb, respectively. 2. Point mutation of plasmid pTMEM43-EGFP-N1containing wildtypeTMEM43gene was introduced via assay of site-directed mutagenesissystem. The mutant TMEM43was identified by restriction enzymedigestion and sequencing. Gel electrophoresis showed a5.7kbfragment be present, and sequencing demonstrated that the pointmutation was successfully introduced.3. The plasmid containing mutant TMEM43(p-mutTMEM43-EGFP-N1)was transformed in competent cells, and then inoculated on selectionplate, pick up positive colonies and to be propagated. The plasmidDNA was prepared by mini-prep kit. Enzyme digestion showed3colonies qualified.4. Sequencing data demonstrated that the correct point mutation C>Tnucleotide replacement was introduced in the TMEM43gene.5. The identified mutant TMEM43expression plasmid was prepared bymaxi-prep kit to carry out a great amount of endotoxin-free plasmidproceeded by the next experiments. The maxi-prepared plasmid wassequenced in whole genomic DNA.6. Cultured HL-1cells which are derived from mouse myocyte cell line,were transfected with/without the recombined eukaryotic expressionplasmid containing wildtype TMEM43(p-wtTMEM43-EGFP-N1) ormutant TMEM43(p-mutTMEM43-EGFP-N1). Under fluorescentmicroscope, transfected cells expressing TMEM43protein. 7. Fluorecent microscopy showed similar localization of both wild typeand mutant expression plasmids in HL-1cells.8. MTT assay demonstrated that growth of cells transfected with wildtype or mutant TMEM43plasmid not significant difference.9. Flow cytometry results showed that the cells transfected with plasmidcontaining mutant (1073C>T) TMEM43behavioured more apoptosisthan those with wild type one.Conclusions:1. The interested point mutation is successfully introduced in thetransmembrane protein TMEM43gene.2. The constructed eukaryotic expression plasmids containing wildtype ormutant TMEM43gene efficiently transfect the cultured myocytes.3. Apoptosis is increased in transfected HL-1cells with the plasmidscontaining mutant TMEM43gene.