| In this study,the CnAβ gene of rat RBL-2H3 cells was knocked out by CRISPR/Cas9 technology to construct a knockout cell mutant.To investigate the effect of CnAβ gene on the activation and degranulation of RBL-2H3 cells in vitro.Three single guide RNAs(sgRNAs)were designed to target exon 1 of the CnAβ gene.A targeting vector expressing sgRNA was constructed using the pX459 plasmid as a backbone.The constructed plasmid was introduced into rat RBL-2H3 cells by liposome 3000 transfection to achieve gene knockout.After puromycin screening,the knockout of the target gene was initially detected by PCR amplification and sequencing.PCR was performed to verify the transcription of three kinds of cells(successfully knocked out cells KO,empty vector cells MOCK,wild cells WT).The morphological changed of rat RBL-2H3 cells after degranulation were detected by toluidine blue staining.The cell viability and toxicity were determined by the WST-1 method.The optimal induction time and dose of the inducer(DNP-BSA,C48/80,LPS)and degranulation of the cells were detected after IgE induction.The main results of this study are listed as follows:1.Using the CRISPR/Cas9 technology,the CnAβ gene of rat RBL-2H3 cells was successfully knocked out.A complete knockout of 381 bp base near exon1 was detected by PCR amplification(exon1 was completely knocked out).2.After PCR amplification of three kinds of cells,gel electrophoresis showed that the knockout cells had no specific bands,indicating that the knockout of the CnAβ gene could not be detected at the transcriptional level.3.After degranulation was induced by A23187,the three kinds of cells were stained with toluidine blue reagent and placed under a 40-fold oil microscope to observe the state.When the cell was degranulated,the cells were dyed in light blue,the cells became larger,the edges of the cell membrane were not aligned,the cytoplasm had obvious particles,and the vacuole structure exists.4.After culturing the three kinds of cells for 5 days in the ratio of medium to WST-1 of 10:1,the growth curve showed that the cells were in the logarithmic growth phase on the first day,and then they were in a stationary phase.And there was no difference in the growth trend of the three kinds of cells,that is,knocking out the CnAβ gene had no effect on cell growth.5.The results of the WST-1 toxicity test showed no difference in cell growth tendency,that is,knocking out the CnAβ gene had no toxic effect on cell growth.6.The optimal induction concentrations of C48/80,DNP-BSA,and LPS were 100 μg/ml,10 μg/ml,and 1 μg/ml,respectively.The optimal induction time of all three was 1 hour.In contrast,DNP and C48/80 were equally capable of inducing degranulation,while LPS was weaker,but significantly reduced cell degranulation after knocking out the CnAβ gene.In summary,cell mutants were successfully constructed by knocking out the CnAβ gene of rat RBL-2H3 cells using CRISPR/Cas9 gene editing technology.Mast cells became large and round after induction of degranulation,and the staining becomes shallow and vacuolar structures appear.The optimal concentrations of cell degranulation induced by the three inducers(C48/80,DNP,LPS)were 100 μg/ml,10 μg/ml,and 1 μg/ml,and the optimal induction time was 1 hour.Knocking out CnAβ gene significantly reduced cell degranulation,but had no effect on cell growth. |
