Study On Key Genes That Regulate Cell Migration Directionality And Speed In Dictyostelium Cells In Electrotaxis

Electrotaxis is a phenomenon in which cells migrate towards the cathode or anode in an electric field.Recent studies have shown that electrotaxis plays an important role in various physiological and pathological events in multicellular organisms.However,researchers still do not fully understand the mechanism of cell electrotaxis.At present,Dictyostelium discoideum as a model organism is often used to study the movement of eukaryotic cells.Because Dictyostelium discoideum cells show obvious electrotactic motion in a DC electric field,they are also used as an ideal model organism for studying cell electrotaxis.After stimulating Dictyostelium cells with electric field,we sequenced its transcriptome and performed preliminary analysis,and obtained some genes whose gene expression was significantly different from that of control cells.In this study,we will knock out the genes with the most significant differences in expression,study the response of gene-edited cells with a DC electric field,and confirm the importance of these genes in cell electrotaxis.Use the following methods to continue experiments,(1)A self-made electrotaxis chamber was constructed and an electric field of 12V/cm was applied to the mutant cells of the Dictyostelium discoideum,and the cell movement was recorded by a realtime video recording system within 30 min,using image J and Usiigaci trackers,the direction and velocity of cell movement were statistically analyzed.(2)Using CRISPR/Cas9 gene editing vector PTM1285 to construct mutant cells of Dictyostelium discoideum,the gene mutants were obtained by electroporation,monoclonal sorting and PCR sequencing.The electrotaxis of the mutant cell lines was studied in the electroporation chamber.The following results are obtained,(1)According to the transcriptome,pi3k1/2,pi3k1-5,sgc A,for C,ras D,erk B,myo A/B/II,mhc A/tal A,pax B,gbp B,gbp C,gbp D,car B,card,Ras Gef D,G?2,G?,G?,pka C,ras C were selected,the result of electrotaxis about these gene mutations showed that these genes can affect electrotaxis in a degree.G?2,G?,myo A/B/II,gbp B,car B and pka C genes influence the direction of cell movement in electric field.sgc A and car D genes influence the velocity of cell movement in electric field.The car B and car D genes will slow down the movement of cells in the electric field.Pi3k1-5,G?2,G?,G?,ras D,pax B,mhc A/tal A,gbp D,erk B,for C,ras C,myo A/B/II,gbp B,car B and pka C will affect the movement direction of cells in the electric field and weaken the directional migration ability of cells to the cathode,but Pi3k1/2 and Ras Gef D will enhance the directional migration ability of cells to the cathode.(2)pik A,pik B,pik C,pik F and pik G gene mutants were constructed by CRISPR/cas9 gene editing technology.Six mutants were obtained by pik A gene,with a total of two mutation types;One mutation type was obtained by pik B gene;pik C gene obtained two mutation types;pik F gene obtained a mutation type;pik G gene obtained three mutation types.(3)The results showed that the deletion of pik A,pik C and pik G genes had little effect on the electrotaxis of Dictyostelium discoideum cells.However,the deletion of pik B gene and pik F gene accelerated the movement speed of the mutant under 12V/cm electric field,and the deletion of pik B gene had a greater effect on the movement speed.In a word,15 genes in the Dictyostelium discoideum are involved in the regulation of speed in electric fields,while,14 genes in the Dictyostelium discoideum are involved in the regulation of direction in a DC electric fields.