Geração de hiPSCs SOX17-RFP e PRDM1-GFP knock-in via CRISPR/Cas9 para estudo da oogênese humana in vitro

Abstract

The generation of hiPSCs containing reporter alleles is an innovative and highly advantageous strategy for the in-depth study of cellular differentiation processes. This study initially aimed to review the scientific literature on the subject and subsequently expand this knowledge through the generation of genetically modified hiPSC lines using the CRISPR/Cas9 system, targeting the expression of fluorescent proteins associated with the transcription factors SOX17 and PRDM1, key markers of primordial germ cell (PGC) development. This approach was designed to enhance the monitoring of differentiation induction protocols in human PGCs, contributing to the development of in vitro models of oogenesis. Initially, we conducted a detailed characterization of the hiPSCs used. These cells exhibited typical morphology, confirmed by light and transmission electron microscopy, and expressed key pluripotency markers, detected by immunofluorescence. The karyotype analysis demonstrated genomic stability with a normal pattern (46, XX). Cell viability assays determined the minimum lethal concentrations of the selective antibiotics blasticidin and puromycin, set at 1.6 µg/mL and 0.8 µg/mL, respectively, which are essential parameters for ensuring efficient post-modification cell selection. The synthesis of donor DNAs was optimized using the inner-outer overlapping PCR technique, which employs pairs of overlapping primers covering internal and external regions of the amplicon. This approach demonstrated greater efficiency and precision compared to the use of long conventional primers. Transfection assays using Cas9-GFP indicated that approximately 26% of hiPSCs internalized the gene-editing complexes one hour after exposure, providing a solid foundation for the targeted integration of SOX17-RFP and PRDM1-RFP. Additionally, we standardized a protocol for definitive endoderm induction, aiming to promote the expression of SOX17 and PRDM1 markers. The validation of this induction was performed through immunofluorescence and qPCR, demonstrating that the employed medium enables rapid and precise activation of the target markers. This strategy presents itself as a practical and efficient alternative to traditional PGC induction methods, which require more time and resources. In parallel, we investigated the effects of valproic acid (VPA) and AZD 7648 treatment on viability, proliferation, and homology-directed repair (HDR) efficiency in knock-in cells. The results indicated that these drugs improve cell viability, although they do not necessarily increase proliferation rates. Moreover, the treatments facilitated the successful generation of knock-in cells for the target markers. As a key outcome, we established two hiPSC lines with fluorescent reporter alleles: SOX17-RFP-Blast and PRDM1-GFP-Puro. The genetic modification was confirmed by definitive endoderm induction, followed by fluorescence microscopy analysis and junctional PCR reactions to detect the insertions at the 5' and 3' ends of the respective genes. The advancements achieved in this study highlight the importance of integrating detailed characterizations, optimized molecular techniques, and well-defined protocols. The generation of hiPSC lines with fluorescent reporter alleles represents a significant step forward in studying the molecular mechanisms governing human oogenesis. In addition to deepening our understanding of the early stages of germ cell development, these lines provide a promising platform for future biomedical research applications.