AbstractsBiology & Animal Science

Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2011 Neural Stem Cells (NSC) are multipotent stem cells, capable of proliferating and differentiating in vivo and in vitro into astrocytes, oligodendrocytes and neurons. For this reason they hold a great potential for the development of gene and regenerative therapies for the treatment of neurodegenerative diseases and brain cancer. However, transfection of NSCs has proven to be difficult through conventional methods, and the disadvantages associated with the use of viral vectors make non-viral vectors more suitable for the development of gene delivery assays to NSCs. Apart from the non-viral method, one of the most important factors in gene delivery is the type of used vector. One of the factors that mostly affect the vector efficiency is the presence of CpG motifs. These motifs are responsible for the triggering of innate and acquired immune responses contributing to episomal silencing of the transgene. In this study, gene delivery to NSCs was optimized for the use of microporation technology and transfection efficiency was compared for the use of different transfection vectors with low vs. high CpG content, namely, minicircles, pCMV-GFP and pVAX-eGFP. The optimization of microporation conditions revealed that depending on the electroporation buffer, high number of transfected cells (60 to 75%) and low cell mortality (15-10%) are obtained when using 1500V, 20 ms and 1 pulse or 1800V, 20 ms and 1 pulse as microporation conditions. When comparing the transfection efficiency using different vectors it was evident that Minicircle was the vector that allowed the obtainment of sustained and higher number of transfected cells (75%) without affecting their survival (80-90% of cell viability) and morphology. The quantification of vector copies in the nuclei revealed that the optimal dose to transfect NSCs is around 0.8 μg, and that, although a similar number of Minicircle and pCMV-GFP copies per nucleus is found, the first are the vectors that yield the highest expression levels. Long term analysis also showed Minicircles are less degraded, exhibiting higher number of copies and GFP expression than pCMV-GFP or pVAX-eGFP. Finally, microporation did not seem to affect NSCs differentiation potential. Taken together, these results offer the first insights in the use of microporation and minicircles in non-viral transfection of NSCs, suggesting that microporation is a promising tool for NSCs transfection and that minicircles offer a new model of efficient and safe non-viral gene delivery to NSCs and have unquestionably a potential use for clinical applications and genetic engineering. Células estaminais representam um grupo específico de células indiferenciadas que apresentam a capacidade de se auto-renovar e de se diferenciar, quando estimuladas por determinadas condições, em células várias linhagens distintas. Existem dois tipos distintos de células estaminais: embrionárias e adultas. As células embrionárias…