REPROGRAMMING OF SKIN FIBROBLAST AND UMBILICAL CORD CELLS THROUGH VIRAL PLASMIDS AND TRANSPOSONS IN THE EQUINE IPS PRODUCTION.

• Grant number: 12/18735-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: December 01, 2013
• End date: November 30, 2016
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Fernanda da Cruz Landim
• Grantee: Mídyan Daroz Guastali
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

In recent decades, research involved on the biology of stem cells has addressed a broad spectrum of phenomena, ranging the tissue and cellular level, until their use in cell therapy. This growing attention suggests that we need to review basic concepts of the organization of stem cells, to understand fully the processes of functional differentiation. Thus, the instrument of cellular reprogramming through the genetic manipulation provides information to better understand the process of renewal and differentiation are fundamental characteristics of stem cells. Although cell reprogramming by viral plasmids has been success reported in several animal species, other alternative techniques can also be employed, such as using transposons, or pieces of DNA that can be introduced into cells and performing the reprogramming eliminating the unwanted viral particle. Researches involving the production of iPS of large animals are still scarce. Thus there is no knowledge of what the best cell type to be used, and neither of which the most efficient method of reprogramming. It is known that the cord has a reservoir rich in mesenchymal stem cells, which to be multipotent may represent an alternative to improve the efficiency of obtaining iPS compared to the use of fibroblasts. The IPSs can potentially be used for regenerative therapy for the study of diseases and drugs and also in improving the results of therapeutic cloning and animal, having a great potential for use in various fields of knowledge including equine medicine. The objective of this project is to obtain iPS via non-viral transfection and viral in umbilical cord cells and fibroblasts horses, for to observe what type of transfection and cell type are more efficient for cellular reprogramming. For both cell types will use transposons and viral plasmids containing genes OCT-4,SOX2, C-MYC and KLF-4 added to the GFP gene so that transduced cells can be identified (GFP positive); evaluate changes caused in the profile cell gene expression by real time PCR; accompany changes in cell equine morphology post-transfection; evaluate the modified cells by immunocytochemistry with the typical markers of embryonic stem cells (such as family and SSEA TRA), and lastly we will induce these cells to differentiate in the tissues of the three germ layers. We expect that the use of transposons and equine umbilical cord cells to increase production efficiency of iPS compared to the traditional method.

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

GUASTALI, Mídyan Daroz. Reprogramming of skin fibroblasts and cord cells by viral plasmids and transposons in the production of equine iPS. 2016. Doctoral Thesis - Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu Botucatu.