Development of species-specific vectors to enhance the efficiency of generating induced pluripotent stem cells in animal models.

• Grant number: 24/13586-8
• Support Opportunities: Scholarships in Brazil - Master
• Start date: February 01, 2025
• End date: August 31, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Fabiana Fernandes Bressan
• Grantee: Nayanne Sant Clair Cardoso da Silva
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil

Abstract

Even though the production and research of iPSCs have gradually advanced and there are attempts to extrapolate the technique to different animal species, there is still a limitation to human and murine organisms as models.Once properly reprogrammed, they present the potential for differentiation in any tissue of the organism, bringing numerous benefits to regenerative and reproductive medicine, as well as leveraging in vitro studies, allowing greater understanding of the biological mechanisms involved in the reprogramming process. However, this process may not be fully carried out given differences in the exogenous gene sequences introduced into the host during the inductive process, in relation to the gene sequence of the species of interest.In this study, we propose the construction of a cell reprogramming induction vector containing species-specific transcription factors (Oct4, Sox2, c-Myc, Lin 28, Nanog and Klf4) with the hypothesis of allowing complete reprogramming and amplifying the efficiency in the acquisition of stem cells in domestic, conventional and unconventional species, the focus of previous research in the country.To this end, gene cloning will be carried out and a lentiviral vector will be assembled that contains genes related to the pluripotency of the species Gallus gallus, and cell culture of fetal chicken fibroblasts, from the commercial lineage DF-1, will be carried out and, subsequently, a characterization of the induced pluripotent cells (iPSC).The construction process of the species-specific induction vector will begin with the extraction of RNA from the target species, followed by cDNA synthesis; polymerase chain reaction for exponential amplification of target sequences; electrophoresis using primers constructed using the species' genomic sequence to reveal whether such segments have the expected base pair size; gel purification to isolate the fragment of interest; Sanger sequencing, to ensure that the nucleotides have the appropriate conformation; linearization of the chosen vector (through restriction enzyme sites) allowing the ligation of fragments (transcription factors) and closure of the vector, which must then be introduced into competent bacteria to observe the formation of bacterial colonies and subsequent isolation of the contents of the plasmid that will be used for viral production.The process of viral acquisition, in turn, containing species-specific factors, is achieved through the use of helpers that provide structural and replication proteins, allowing viral packaging with content of interest, which will be delivered to host cells 293 FT, which they will provide the cellular machinery necessary for the replicative process and will allow the formation of countless viral copies that will be ultra-centrifuged and concentrated so that they can be translated into adult cells and induce the return of the pluripotency state similar to the embryonic one.Therefore, success in assembling a vector containing specific sequences of chicken cDNAs related to genes related to the induction of pluripotency, namely OCT4, SOX2, c-myc, KLF4, Lin28 and Nanog, is expected. Furthermore, the vector must be functional, which will be tested for its cell reprogramming capacity when compared to the vector already commercially available, with human cDNAs.Furthermore, it is expected to promote the expansion of knowledge of the cellular and molecular aspects of pluripotent cells for subsequent application of this technology to improve cellular studies of other species of interest to the research group. This experience will be essential for optimizing current protocols for complete reprogramming in animal cells, including greater efficiency compared to in vitro induced reprogramming (generation of iPSCs) from models other than rodents and humans.