Mimicking embryonic interactions with engineered cell-culturing techniques
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Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2017
Författare
Obermüller, Jakob
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
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 [1]. 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.[1]. 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.
Beskrivning
Ämne/nyckelord
Livsvetenskaper , Biologiska vetenskaper , Biokemi , Life Science , Biological Sciences , Biochemistry