The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhan...The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.展开更多
The P19CL6 cell-line, a clone of the P19 mouse embryonal carcinoma cell-line, has been exten-sively used as a model for cardiomyocytes as these cells can be differentiated into a cardio-myocyte phenotype upon incubati...The P19CL6 cell-line, a clone of the P19 mouse embryonal carcinoma cell-line, has been exten-sively used as a model for cardiomyocytes as these cells can be differentiated into a cardio-myocyte phenotype upon incubation with di-methyl sulfoxide. Uniquely, these cells can be observed to “beat” when monitored by mi-croscopy. We started investigating the response of P19CL6 cells to fatty acids, but highly vari-able results lead us to investigate the phenotype of the P19CL6 cells in more depth. In this study we demonstrated that the P19CL6 cells are re-sponsive to adrenaline, but loose the “beating” phenotype after 16 passages in culture. Analysis of specific mRNA transcripts indicated that the P19CL6 cells express both cardiac- and skeletal muscle-specific genes, while global analysis of microarray data showed clear differences be-tween the P19CL6 cells and cardiac tissue of embryonic or adult origin. In conclusion, our observations suggest caution in the use of the P19CL6 cells as a model of cardiomyocytes unless rigorous validation for the intended analysis has been undertaken.展开更多
文摘The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.
文摘The P19CL6 cell-line, a clone of the P19 mouse embryonal carcinoma cell-line, has been exten-sively used as a model for cardiomyocytes as these cells can be differentiated into a cardio-myocyte phenotype upon incubation with di-methyl sulfoxide. Uniquely, these cells can be observed to “beat” when monitored by mi-croscopy. We started investigating the response of P19CL6 cells to fatty acids, but highly vari-able results lead us to investigate the phenotype of the P19CL6 cells in more depth. In this study we demonstrated that the P19CL6 cells are re-sponsive to adrenaline, but loose the “beating” phenotype after 16 passages in culture. Analysis of specific mRNA transcripts indicated that the P19CL6 cells express both cardiac- and skeletal muscle-specific genes, while global analysis of microarray data showed clear differences be-tween the P19CL6 cells and cardiac tissue of embryonic or adult origin. In conclusion, our observations suggest caution in the use of the P19CL6 cells as a model of cardiomyocytes unless rigorous validation for the intended analysis has been undertaken.
