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2012.igem.org:Wlc
From 2012.igem.org
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Project
Our team (the Wisconsin Lutheran College iGEM team) is designing a tool that will aid researchers in their study of stem cell differentiation. The iDifferentiate “Operating System” uses genetic engineering to select for cells of a certain lineage. The “App” that we have designed, the SAVE Assay, focuses on selection and isolation of atrial and ventricular cells.
Our main goal for the 2012 iGEM competition is to synthesize two plasmids. We selected two promoters, one that is atrial-specific (of the gene ANF) and one that is ventricular-specific (of the gene MLC2v). The ANF promoter will drive the transcription of eGFP, so only atrial cells will express eGFP. The MLC2v promoter will drive the transcription of eCFP, so only ventricular cells will express eCFP.
Furthermore, the stem cells containing both plasmids will receive puromycin and neomycin resistance. This will be achieved by the use of a constitutive promoter driving expression of the antibiotic. The puromycin resistance will be conferred via the plasmid containing the ANF-eGFP construct, and neomycin through the plasmid with the MLC2v-eCFP construct. This allows us to select for stem cells that contain both plasmids by adding neomycin and puromycin to our growth medium. The SAVE Assay facilitates study of the differentiation pathway between ventricular and atrial cardiomyocytes. The ability to analyze changes in the ratio and abundance of ventricular and atrial cardiomyocytes will be extremely useful in many research and treatment applications.
Our secondary goal for the 2012 iGEM competition is to engineer a feeder layer consisting of fibroblast cells. The purpose is to provide a matrix on which to grow and receive nourishment. Furthermore, the feeder layer will be neomycin and puromycin resistant. This way, the fibroblast cells will not be harmed by the neomycin and puromycin for selection of atrial and ventricular cells.
