Germline Transgenic Methods for Tracking Cells and Testing Gene Function During Regeneration in the Axolotl

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum(axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible

Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

Peer reviewedPublisher PD

Characterization of Sirt2 using conditional RNAi in mice

Within the past eight years, RNA interference (RNAi) has emerged as a powerful experimental tool for gene function analysis in mice. Reversible control of shRNA mediated RNAi has been achieved by using a tetracycline (tet)-inducible promoter. In the presence of the inductor doxycycline (dox), shRNA mediated gene silencing is initiated, whereas RNAi mechanism is blocked in the absence of dox. To achieve spatially and temporally regulated RNAi, the tet inducible system was combined with a Cre/loxP based strategy for tissue specific activation of shRNA constructs. To this end, a loxP-flanked "promoter inhibitory element" (PIE) was placed between the proximal (PSE) and distal sequence element (DSE) of a dox inducible promoter such that promoter function is completely blocked. Re-activation can be achieved through Cre mediated excision of PIE. To allow for gene silencing in a selected tissue, Cre expression can be regulated by a tissue-specific promoter. In mouse ES cells, the system mediated tight regulation of shRNA expression upon Cre mediated activation and dox administration, reaching knockdown efficiencies of >80%. Unexpectedly, the system showed a limited activity in transgenic mice when applied for conditional silencing of two different targets, LacZ and Sirt2. Sirt2 is a member of the sirtuin family which has considerably gained attention in vitro for its possible role in many physiological processes, including adipogenesis and neurodegenerative diseases. To investigate the function of Sirt2 in vivo, the unmodified dox-responsive and tet-inducible promoter was further used for conditional RNAi in transgenic mice. Inducible shRNA expression resulted in efficient silencing of Sirt2 (>90%) in all tissues which have been analyzed. Suppression of Sirt2 during embryogenesis resulted in offspring consisting of equal ratios of wild type and transgenic pups, indicating that Sirt2 is not indispensable for development. In adult animals, glucose metabolism, insulin sensitivity and energy balance appeared to be unaffected by Sirt2 deficiency. Likewise, expression of PPARγ, a downstream target of Sirt2, was not found to be altered upon Sirt2 inhibition. Finally, Sirt2 silencing was induced in an experimental model of Parkinson disease (PD). Data from Rotarod performances to study motor behaviour did not provide any evidence for a role of Sirt2 in PD pathogenesis as suggested by previous in vitro studies. Taken together, conditional Sirt2 silencing in vivo does not support speculation concerning a central role of Sirt2 in physiological processes, embryogenesis and in a mouse model of Parkinson disease

Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease

Generation of inducible Cre systems for conditional gene inactivation in mice

Inducible gene targeting based on the Cre/loxP system is a novel technique allowing conditional gene modification in mice. Precondition of this method is the regulation of Cre activity by inducible genetic switches. This study aimed at improving inducible systems for the regulation of Cre activity at both the transcriptional and the posttranslational level, which should be tested first in vitro and subsequently in newly generated transgenic mice. In order to detect Cre activity, a special double indicator consisting of DsRed and eGFP was constructed and targeted into the ROSA26 locus of HM-1 ES cells. This indicator should constitutively express the DsRed gene flanked by loxP sites. Cre-mediated recombination excises the DsRed gene and turns on the eGFP gene, as evidenced by a change in fluorescence from red to green. Though the double indicator worked properly in transiently transfected fibroblasts, the targeting strategy used for the integration of the indicator into the ROSA26 locus presumably disrupted a putative stem loop motif of the 5'UTR resulting in a silencing of the indicator at the protein level. However, the ES cell line with the integrated double indicator is suited for targetings of inducible Cre constructs into the HPRT locus, since inducibilities are detectable by Southern Blot analysis. The Cre*PR system was one inducible system I have established. In contrast to the classical CrePR1 system, the background activity of Cre*PR was almost abolished by introducing a point mutation into the 3'end of the Cre part, thus preventing aberrant splicing of the Cre*PR encoding mRNA. Also, the PR-LBD of the Cre*PR system was improved, compared to the CrePR1, to obtain a higher affinity towards the inducer RU486. The Cre*PR system, driven by either the EF1£\ promoter or the CAGGS promoter, was then targeted into the HPRT locus. The CAGGS Cre*PR worked in ES cells as expected. These targeted ES cells were subsequently used to generate transgenic mice. In these mice, high protein levels of Cre*PR have to be obtained in order to achieve inducible Cre-mediated excision of loxP-flanked gene segments. In addition, I generated a novel B cell specific Cre*PR mouse strain by targeting Cre*PR into the mCD19 locus. In another approach to inducibly regulate Cre, a tet-on system was established consisting of the rtTA-M2 transactivator and the tetresponsive Cre cassette, which was targeted into the HPRT locus of the double indicator ES cell line. This system revealed a tight regulation of Cre expression in a doxycycline dependent manner

Bacterial artificial chromosomes improve recombinant protein production in mammalian cells

<p>Abstract</p> <p>Background</p> <p>The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production.</p> <p>Results</p> <p>In this work, we explore the efficacy of a Bacterial Artificial Chromosome based vector applied to production of the constant region of the human IgG1. Direct comparison of bulk HEK 293 cell cultures generated with a "conventional" vector or with a BAC-based vector showed that the BAC-based vector improved the protein yield by a factor of 10. Further analysis of stable cell clones harboring the BAC-based vector showed that the protein production was directly proportional to the number of integrated BAC copies and that the protein production was stable for at least 30 passages.</p> <p>Conclusion</p> <p>Generation of stable cell clones for protein production using Bacterial Artificial Chromosomes offers a clear advantage over the use of conventional vectors. First, protein production is increased by a factor of 10; second, protein production is stable overtime and third, generation of BAC-based expression vectors does not imply a significant amount of work compare to a conventional vector. Therefore, BAC-based vectors may become an attractive tool for protein production.</p

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

<p>Abstract</p> <p>Background</p> <p>The <it>Sleeping Beauty </it>(<it>SB</it>) transposon system has been used for germline transgenesis of the diploid frog, <it>Xenopus tropicalis</it>. Injecting one-cell embryos with plasmid DNA harboring an <it>SB </it>transposon substrate together with mRNA encoding the <it>SB </it>transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that <it>SB </it>transposons stably integrated into the frog genome are effective substrates for remobilization.</p> <p>Results</p> <p>Transgenic frogs that express the <it>SB</it>10 transposase were bred with <it>SB </it>transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the <it>SB </it>transposons. The remobilized <it>SB </it>transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'.</p> <p>Conclusions</p> <p>In this study, we demonstrate that <it>SB </it>transposons integrated into the <it>X. tropicalis </it>genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.</p

Single copy shRNA configuration for ubiquitous gene knockdown in mice

RNA interference through the expression of small hairpin RNA (shRNA) molecules has become a very promising tool in reverse mouse genetics as it may allow inexpensive and rapid gene function analysis in vivo. However, the prerequisites for ubiquitous and reproducible shRNA expression are not well defined. Here we show that a single copy shRNA-transgene can mediate body-wide gene silencing in mice when inserted in a defined locus of the genome. The most commonly used promoters for shRNA expression, H1 and U6, showed a comparably broad activity in this configuration. Taken together, the results define a novel approach for efficient interference with expression of defined genes in vivo. Moreover, we provide a rapid strategy for the production of gene knockdown mice combining recombinase mediated cassette exchange and tetraploid blastocyst complementation approaches

The role of Notch signaling in thymic epithelium development

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2011The thymus generates central immune tolerance by producing self-restricted and self-tolerant T-cells as a result of interactions between developing thymocytes and thymic epithelial cells (TECs). While the functional importance of TECs is well established, the mechanisms that direct their embryonic development are unclear. The Notch pathway is a major signaling pathway involved in cell-fate determination. Recently, H. Neves group observed that, during chicken embryogenesis, Notch signaling-related molecules are expressed in the endoderm of the pharyngeal pouches, prior to their specification into TECs, suggesting the involvement of Notch signaling in this process. In this work we aimed to study the role of Notch signaling in early stages of thymic development in chicken. To modulate Notch signaling in vivo, two new plasmids were generated with either the constitutively active form of Notch1 (intracellular domain of Notch1, ICN1) (pT2K-ICN1eGFP), or the dominant-negative (DN) form of Mastermind-like1 (MAML1) (transcriptional co-activator Notch signaling) (pT2K-DNMAML1eGFP). pT2K-ICN1eGFP and pT2K-DNMAML1eGFP were integrated in a “Tol2-mediated gene transfer” and “Tet-On” combined system of vectors developed by Y. Takahashi and collaborators, allowing the study of gain- and loss-of-function of Notch signaling, respectively. Our results show that pT2K-DNMAML1eGFP plasmid in this system, is capable of blocking Notch signaling. Conversely, further assays are required to confirm the functionality of pT2K-ICN1eGFP plasmid. Future studies of gain- and loss-of-function of Notch signaling in early thymic development will be performed by the genetic modification of isolated endodermal tissues of the presumptive territory of TECs with this system. The manipulated tissues will then be grafted into the body wall of a chicken embryo and thymic development followed by in situ observation. In vitro assays of pharyngeal region explants of E3.5 and E4 chicken embryos were performed to study the effect of Notch signaling inhibition during epithelial-mesenchymal interactions in early thymic development. Thymus and parathyroid glands development was assessed by Foxn1 and Gcm2 expression, respectively. We observed that Notch signaling inhibition by a γ-Secretase inhibitor (DAPT) interferes in Foxn1 expression in an apparent random fashion, making inconclusive its role in early thymic development. On the other hand, Gcm2 expression is down-regulated when Notch signaling is inhibited. This data suggests that Notch signaling is required in early stages of parathyroid development. Further studies are essential to unravel the role of Notch signaling in early thymic development.O timo gera tolerância imunitária ao produzir células-T “auto-restritas” e “auto-tolerantes”, cuja geração depende das interacções das suas células precursoras, os timócitos, com células especializadas do nicho tímico, as células epiteliais tímicas (CET). Embora a importância funcional das CET esteja bem estabelecida, os mecanismos moleculares responsáveis pelo seu desenvolvimento embrionário são ainda desconhecidos. O desenvolvimento do timo é acompanhado de perto pelo desenvolvimento das glândulas paratireóides. Os domínios presuntivos do timo e das glândulas paratireóides foram identificados na endoderme da 3ª e 4ª bolsas faríngicas pela expressão de Foxn1 e Gcm2, respectivamente. A especificação das CET depende de interacções entre a endoderme da 3ª e 4ª bolsas faríngicas e o mesênquima circundante - interacções epitélio-mesenquimais. Algumas destas interacções epitélio-mesenquimais começam agora a ser reveladas, nomeadamente através do trabalho do grupo de H. Neves, que demonstra que factores de transcrição como o Bmp4 e o Fgf10 são expressos sequencialmente no mesênquima e são essenciais para a especificação da endoderme da 3ª e 4ª bolsas faríngicas em CET. Outras vias de sinalização têm sido sugeridas como prováveis intervenientes neste processo. A sinalização Notch é uma delas, sendo uma via altamente conservada no reino animal, envolvida nos processos de decisão do destino celular, no desenvolvimento embrionário e no adulto. Esta via de sinalização regula vários processos biológicos, incluindo a hematopoiese, miogénese, neurogénese, vasculogénese, desenvolvimento da pele e outros processos de organogénese. Em 2001, o grupo de L. Parreira descreveu pela primeira vez, num contexto de um nicho estromal, a importância da formação de um microambiente Notch para a correcta especificação de progenitores hematopoiéticos nas diferentes linhagens linfóides. Este e outros grupos também observaram que os genes envolvidos na sinalização Notch são expressos de forma distinta nos diferentes territórios do timo adulto, reforçando a importância desta via de sinalização na função do mesmo. Recentemente, o grupo de H. Neves também observou, em embriões de galinha, que os genes envolvidos na sinalização Notch (receptores, ligandos e genes-alvo) estão diferencialmente expressos na endoderme das bolsas faríngicas, em estádios prévios à formação do rudimento tímico. Também observaram, usando um novo sistema in vitro de associações heteroespecíficas de tecidos embrionários, que a sinalização Notch interfere com a expressão normal de Foxn1 (marcador de CET) na endoderme das bolsas faríngicas. Este sistema de cultura permite uma análise funcional das moléculas envolvidas nas interacções epitélio-mesenquimais e também determinar a dinâmica temporal destas moléculas durante o desenvolvimento do timo. Este sistema recapitula in vitro os acontecimentos precoces do desenvolvimento tímico in vivo, uma vez que, quando enxertadas na membrana corioalantóide do embrião de galinha, estas associações de tecidos desenvolvem um timo funcional. Estes dados sugerem que a sinalização Notch está envolvida na especificação da endoderme das bolsas faríngicas em epitélio tímico. Neste projecto, tivemos como objectivo estudar a sinalização Notch na especificação da endoderme das bolsas faríngicas em epitélio tímico. Para isso, desenvolvemos estratégias in vivo e in vitro de ganho e perda-de-função da sinalização Notch em embriões de galinha. Para a abordagem in vivo, dois plasmídeos novos foram construídos, para expressar a forma constitutivamente activa de Notch1 (domínio intracelular de Notch1, ICN1) (pT2K-ICN1eGFP) ou a forma dominante-negativa de Mastermind-like1 (MAML1) (co-activador transcricional de sinalização Notch) (DNMAML1) (pT2K-DNMAML1eGFP). Estes plasmídeos foram integrados num sistema de vectores desenvolvido por Y. Takahashi e colaboradores, que combina a “transferência génica mediada por Tol2” e a “expressão condicional dependente de tetraciclina”. Assim, este sistema de vectores oferece uma abordagem experimental única para uma análise temporal, e específica de tecido, dos efeitos de ganho e perda-de-função da sinalização Notch durante a especificação/diferenciação das CET. Para realizar esses estudos, células endodérmicas do território gerador do epitélio tímico serão modificadas geneticamente com os plasmídeos pT2K-ICN1eGFP ou pT2K-DNMAML1eGFP (neste sistema de vectores) para activar ou bloquear a sinalização Notch, respectivamente, e serão enxertadas na parede do embrião de galinha, para acompanhar in situ o desenvolvimento do timo quimérico. Os nossos resultados mostram que o plasmídeo que expressa a forma DNMAML1, pT2K-DNMAML1eGFP, neste sistema de vectores, tem a capacidade de inibir a sinalização Notch. O trabalho poderá assim prosseguir para os estudos de perda-de-função da sinalização Notch na endoderme das bolsas faríngicas, antes da sua especificação em CET. Por outro lado, o plasmídeo que expressa o ICN1, pT2K-ICN1eGFP, neste sistema de vectores, necessita de mais experimentação para comprovar a sua funcionalidade. A abordagem in vitro consistiu na realização de ensaios de cultura de explantes da região faríngica de embriões de galinha com 3.5 e 4 dias de desenvolvimento para estudar o efeito da inibição farmacológica da sinalização Notch (com um inibidor da γ-Secretase – DAPT) durante as interacções epitélio-mesenquimais no desenvolvimento inicial do timo. Tanto a especificação da endoderme faríngica em epitélio tímico como a manutenção do domínio das glândulas paratireóides nos explantes em cultura foram avaliadas através da expressão in situ de Foxn1 e Gcm2, respectivamente. Os nossos ensaios in vitro revelaram que a inibição da sinalização Notch, em fases iniciais do desenvolvimento tímico, interfere com a expressão de Foxn1 de forma aparentemente aleatória, mostrando elevada heterogeneidade de resultados. Assim, o papel da sinalização Notch em fases iniciais do desenvolvimento do timo continua por precisar. Por outro lado, a expressão de Gcm2 é bloqueada com a inibição da sinalização Notch nessas mesmas fases de desenvolvimento. Portanto, os nossos dados in vitro sugerem um papel da sinalização Notch no desenvolvimento das glândulas paratireóides, pelo menos em fases iniciais do desenvolvimento. Novas experiências in vitro usando explantes da região faríngica de embriões de galinha em estádios de desenvolvimento diferentes (tanto mais precoces como mais tardios) daqueles estudados, e a realização dos estudos in vivo com o nosso sistema de vectores, serão essenciais para compreender estes resultados e também para estudar o papel da sinalização Notch na especificação/desenvolvimento das CET. Com este projecto esperamos contribuir para o conhecimento do papel da sinalização Notch no desenvolvimento normal das células epiteliais tímicas, um passo fundamental na compreensão dos eventos responsáveis pela manutenção de um timo saudável ao longo da vida e pela reparação da sua função em situações patológicas. Mais, o conhecimento de elementos chave envolvidos na especificação das CET poderá no futuro permitir a criação de novos sistemas in vitro para gerar células epiteliais tímicas, as quais, por sua vez, poderão abrir novas possibilidades de produção in vitro de repertórios de células-T e novas oportunidades para restaurar a função tímica em indivíduos atímicos ou imunodeficientes e, também, melhorar as terapias de transplantação de órgãos

Slingshot: a PiggyBac based transposon system for tamoxifen-inducible ‘self-inactivating’ insertional mutagenesis

We have developed a self-inactivating PiggyBac transposon system for tamoxifen inducible insertional mutagenesis from a stably integrated chromosomal donor. This system, which we have named ‘Slingshot’, utilizes a transposon carrying elements for both gain- and loss-of-function screens in vitro. We show that the Slingshot transposon can be efficiently mobilized from a range of chromosomal loci with high inducibility and low background generating insertions that are randomly dispersed throughout the genome. Furthermore, we show that once the Slingshot transposon has been mobilized it is not remobilized producing stable clonal integrants in all daughter cells. To illustrate the efficacy of Slingshot as a screening tool we set out to identify mediators of resistance to puromycin and the chemotherapeutic drug vincristine by performing genetrap screens in mouse embryonic stem cells. From these genome-wide screens we identified multiple independent insertions in the multidrug resistance transporter genes Abcb1a/b and Abcg2 conferring resistance to drug treatment. Importantly, we also show that the Slingshot transposon system is functional in other mammalian cell lines such as human HEK293, OVCAR-3 and PE01 cells suggesting that it may be used in a range of cell culture systems. Slingshot represents a flexible and potent system for genome-wide transposon-mediated mutagenesis with many potential applications