Mimicking embryonic interactions with engineered cell-culturing techniques
Examensarbete för masterexamen
Biotechnology (MPBIO), MSc
Compaction of the mouse embryo is triggered by the formation of filopodia by some of the blastomeres. These finger-like processes extend onto neighboring cells, provid-ing mechanichal tension and possibly sending a signal that mediates compaction . To investigate whether filopodia-mediated contact induces a transcriptional response in the receiving cells, NIH3T3 murine embryonic fibroblasts were used to design a model system for compaction. Two di˙erent cell-cultures were generated from the fibroblasts by inducing filopodia-formation in one culture (filopodia-expressing cells: FECs) and by adding a membrane marker to the other (non-expressing cells: NECs), allowing for separation on a column. These populations were to be co-cultured to allow filopodial contact to be established between them, after which the contact-receiving cells were to be isolated. The transcriptome of the filopodia-receiving cells would then be characterized. Induction of filopodia could not be achieved by trans-fecting the fibroblasts with Egfp-Myo10 a method used by Fierro-Gonzaléz et al.. However, transfection of Gfp-Cfl1 -constructs encoding GFP-cofilin resulted in filopodia-formation. This provided a reliable method to generate FECs from the fibroblasts. Moreover, an NEC cell-line that stably expressed the membrane tag Vamp2-Sbp was generated using antibiotic resistance on the Vamp2-Sbp plasmid. Collectively, these results show that the underlying mechanisms behind filopodia-formation may vary, depending on cell-type and environmental parameters such as substrate composition. Furthermore, the findings provide a firm base upon which to build a model system to further study the intercellular filopodial contact in mam-malian cells.
Livsvetenskaper , Biologiska vetenskaper , Biokemi , Life Science , Biological Sciences , Biochemistry