GENERATION OF REPORTER CELL LINES TO STUDY THE DINAMICS OF INTERACTIONS OF THE TRANSCRIPTION FACTORS OCT4 AND SOX2 WITH THE CHROMATIN DURING CELL REPROGRAMMING

SOLARI C; PETRONE MV; ALBERIO RH; COSENTINO S; VERNERI P; CANIZO J; GUBERMAN A; VAZQUEZ ECHEGARAY C; OSES C; FRANCIA M; LEVI V

Ciudad de Buenos

Congreso; Reunión Conjunta de Biociencias 2017; 2017

Due to the lack of success to obtain genuine induced pluripotent stem cells (iPSCs) in some species like farm animals, with a biotechnology interest, we decided to focus our study in the reprogramming process itself using a well characterized model as murine cells. It is well known that the remodeling of the chromatin and the core pluripotency transcription factors (TFs) Oct4, Sox2 and Nanog, are key elements during cell reprogramming. For this purpose, we generated doxycycline-inducible stable cell lines over-expressing Oct4 or Sox2 C-terminally fused to a yellow fluorescent (YPet) by lentiviral transduction of the cell lines NIH3T3 (mouse fibroblasts), W4 (mouse embryonic stem cells) and IPS17 (mouse iPSCs generated in our lab). We analyzed the different individual clones by studying the morphology and cell cycle with propidium iodide and cytometry, and selected those which presented normal phenotype in the absence of doxycycline, using as control the parental cell lines. To test the ?Tet-on? system we tried a dose curve of doxycycline in the generated cell lines, and selected a 2 ug/ul concentration to perform the upcoming experiments. On the contrary, we analyzed the time required to shut down the expression of YPet-TF, established in 48 hrs. Moreover, we studied the functionality of the transgenes, after induction, by immunofluorescence and RT-qPCR examining the expression of Oct4, Sox2 and Nanog under the different scenarios. We are currently setting the conditions to study the obtained cell lines using confocal microscopy in fluorescence correlation spectroscopy (FCS) experiments, which will reveal the distinct dynamics of interactions between the TFs and the chromatin, visualized by the expression of the chromatin reporter fusion protein H2B-RFP. Overall, we obtained a powerful tool to study the complex process of cell reprogramming by different approaches and to contribute to elucidate the intricate dynamics that could be taking place in refractory species.