CONSTRUCTION OF AN EMBRYONIC STEM CELL LINE BY CRISPR/CAS9 MEDIATED HOMOLOGOUS RECOMBINATION TO STUDY THE DIFFERENTIATION DYNAMICS IN LIVING CELLS

JUAN PURACCHIO; RODRIGO SKOP; FEDERICO SEVLEVER; ANTONELLA LOMBARDI; LUCÍA MORO; SANTIAGO MIRIUKA; ARIEL WAISMAN

Buenos Aires

Congreso; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2021

Mouse embryonic stem cells (mESCs) are pluripotent cells that resemble the preimplantation epiblast of the mouse embryo. Theycan give rise to all the cells of the organism, making them an excellent model to study mammalian development in vitro, as well as a cell type of great interest in regenerative medicine. Stem cell differentiation is a highly dynamic process in which epigenetic, transcriptional and metabolic changes eventually lead to new cell identities. These modifications occur within hours to days and are generally identified by measuring gene expression changes and protein markers. Transcription factor Oct6, encoded by Pou3f1gene, is a protein expressed during the early phase of mESCs differentiation. Previous results from our laboratory have shown that the up regulation of Oct6 remarkably affects the expression of NANOG, a key transcription factor that helps mESCs maintain pluripotency. Although immunofluorescence of fixed cells is the standard technique to allow the visualization of these proteins in individual cells, this method prevents the analysis of dynamical processes in living cells. In the present work, we aimed to generate a reporter line of mESCs differentiation by fusing the endogenous Oct6 protein together with mCherry, a red fluorescent protein. For that, we used CRISPR-Cas9 technology to achieve a highly precise and efficient knock-in through homologous recombination. We designed an antibiotic-selection based strategy in which the fluorescent reporter, plus a small flexible glycine linker, is introduced at the Oct6 C terminus, creating a new Oct6-mCherry fusion protein product. The targeting vector was promoterless, and consequently mCherry signal can only arise from the correct, in-frame insertion at the endogenous Oct6 locus in differentiating cells. This vector, which was generated through DNA synthesis, included multiple restriction sites to allow the future targeting of different genes, cloning of other reporter proteins and utilized LoxP sequences flanking the G418 resistance to subsequently remove it through CRE recombinase-mediated recombination. We cloned two different sgRNA targeting the STOP codon of Oct6 into a CRISPR/Cas9 plasmid and transfected them into mESCs together with the targeting vector. A high proportion of the G418 resistant cells displayed mCherry fluorescence after inducing cell differentiation, indicating the correct insertion of the reporter cassette. We next isolated 9 clonal cell lines for further characterization, of which 8 displayed mCherry fluorescence in differentiating cells. Western blot analysis of these clonal lines confirmed that the mCherry expressing cells were heterozygous for the incorporated fluorescent protein, while the non-fluorescent clone only expressed the wild type alleles. Once fully characterized, this recombinant cell line will provide significant new opportunities for live-cell imaging to study the cellular and molecular dynamics of mESCs differentiation