Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

Livro de resumos do IV colóquio nacional de horticultura biológica

A agricultura biológica tem futuro e o futuro é hoje! É o mote do IV Colóquio Nacional de Horticultura Biológica, que decorre de 17 a 19 de março, no Anfiteatro Verde da Universidade do Algarve, em Faro. Uma iniciativa da Associação Portuguesa de Horticultura (APH) em parceria com a Universidade do Algarve e a Direção Regional de Agricultura e Pescas do Algarve. O IV Colóquio Nacional de Horticultura Biológica pretende ser um espaço de troca de conhecimento, de experiências e de debate que contribua para demonstrar o potencial produtivo, económico e ambiental da agricultura biológica (AB). Este modo de produção agrícola tem vindo a crescer na União Europeia nos últimos 10 anos a uma taxa média anual de 8% em área e de 11,6% em mercado, ultrapassando em 2012 os 20 000 milhões de euros em vendas. No mesmo ano, a AB ocupava em Portugal 226 425 hectares, distribuídos por 2885 produtores, principalmente pelo Alentejo e Beira Interior e em Trás-os-Montes, com predomínio das pastagens e do olival. A área de hortofrutícolas e de vinha em modo de produção biológico (MPB) é muito baixa, devido a maior dificuldade técnica e pela falta de experimentação e divulgação nestas culturas, mas é um potencial de produção e valorização agrícola que o país deve aproveitar. A agricultura em MPB encontra-se num contexto difícil, com alterações legislativas a nível europeu mais restritivas e a premente necessidade de reduzir custos de produção. É também indispensável fomentar o aumento da produção (novos produtores) e aumentar o valor dos produtos agrícolas biológicos através do seu processamento local. A certificação rigorosa e credível é indispensável para manter a confiança dos consumidores. A resposta a estes problemas deve ser encontrada através da inovação e partilha da informação entre produtores, técnicos e investigadores, de forma a tornar a agricultura biológica mais competitiva, aumentando o emprego e fortalecendo a economia rural, sendo este o intuito do IV Colóquio Nacional de Horticultura Biológica.