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.

Gene activation and re-expression of a trypanosoma-brucei variant surface glycoptotein

The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so-called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site-transposon junction, and the 5' half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co-transposed segment 5' to the coding sequence is 430 bp in length. The extreme 5' end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.

"A beleza é o grau mais elevado da verdade", "Os Memoráveis", de Lídia Jorge

Foi um prazer ler o último romance de Lídia Jorge, editado em março último, pela Dom Quixote. E as razões foram muitas. Porque fala de um dia da nossa história que me diz bastante: o 25 de abril de 1974. Apesar de ter dele apenas uma vaga ideia, foi sendo sempre falado na minha família e faz parte do meu presente. Porque reconheço grande parte da história ali contada, fazendo-me sentir cúmplice, quer do texto, quer dos acontecimentos. Porque o romance é um género que faz falta para contar a História. É um modo de chegar a muito mais gente que, depois de o ler (ou enquanto o vai lendo), vai ter vontade de ir procurar os outros livros – os de História não romanceada – para aprender sobre as horas daquela noite de 24 para 25 e sobre os seus protagonistas. Apesar da «transfiguração literária », como se lê na nota de edição, quem sabe se não os reconhecerá? E saltando muitas outras razões, porque é um livro muito bem escrito. As pontas que vão sendo soltas ao longo da narrativa juntam-se em outros momentos, completando quadros de sentido. Ana Maria Machada, a narradora, como participante da história, sabe tanto como nós sobre o que pensam as outras personagens, mas sabe um bocadinho mais do que, em certos momentos, conta. Por exemplo, quando a equipa de reportagem entrevista a viúva de um dos capitães de Abril (que percebemos ser Salgueiro Maia, apesar de apenas ser referido pela sua «alcunha doméstica», isto é, pelo nome que a mãe de Ana Maria lhe dera: Charlie 8) e tenta conseguir que esta diga quem queria mal ao marido, perante a relutância em acusar alguém, a «Machadinha» afirma «Nós sabíamos, mas não tão bem como ela, que as vinganças de que foram vítimas ele e os outros como ele, tinham tido autores concretos, nomeáveis, intérpretes e responsáveis, colocados no topo das estruturas criadas num país onde passara a haver liberdade para legitimar tudo e o seu contrário» (p. 249).