Die einzellige Grünalge C. reinhardtii dient als Modellorganismus und ist aufgrund des GRAS-Status, des schnellen, kostengünstigen Wachstums und der Möglichkeit posttranslationaler Modifikationen im Kerngenom, als Expressionssystem für beispielsweise orale Impfstoffe sehr interessant. Herausforderungen sind die im Vergleich zu konventionell verwendeten Expressionssystemen sehr geringen Expressionsraten im Kerngenom. Daher sollten in dieser Arbeit neuartige, teils induzierbare, Promotorkonstrukte verwendet und mittels Luciferase-Reporter auf ihre Expressionssteigerung hin getestet werden. Zunächst wurden ausgewählte Promotoren des Chlorella-Virus-1 (PBCV-1) gewählt, diese führten allerdings zu keiner Expression. Außerdem wurden synthetische, aneinandergereihte Hitzeschockelemente mit dem endogenen RBCS2-Promotor fusioniert und die Expressionsraten analysiert. Dabei ergab sich bei der Kombination aus dem synthetischen Hitzeschockelement in achtfacher Wiederholung (HSE8x) mit RBCS2 nach der Hitzeinduktion eine Steigerung der Expressionsrate um das bis zu dreifache. Die Basalexpression war hierbei bei HSE1x-RBCS2 am höchsten und erreichte Expressionslevels, welche um das fünffache höher lagen als die Positivkontrolle HSP70A-RBCS2. Mittels Chromatinimmunopräzipitation mit dem Antikörper gegen HSF1 konnte gezeigt werden, dass eine Bindung an das synthetische Hitzeschockelement vorliegt und deshalb die Expression über die konventionelle Hitzeschockantwort in Chlamydomonas reinhardtii funktioniert. Im Gegensatz zur Luciferase benötigen Fluoreszenzproteine als Reporter kein Substrat. Infolge dieses Vorteils und der Möglichkeiten der FACS-Analyse und Fluoreszenzmikroskopie wurde tagRFP als neuer Reporter in C. reinhardtii etabliert. Die Erhöhung der Expressionsrate durch neue Promotorkombinationen und die Anwendung von tagRFP als neuen Fluoreszenzreporter bedeuten wichtige Schritte in der Etablierung von C.reinhardtii als Expressionssystem für Produkte in der Biotechnologie. / The unicellular green alga C.reinhardtii which is used as a model organism could be an interesting expression system for oral vaccines. This is because the alga is generally regarded as safe, it shows fast growth rates and culturing is cheap. Furthermore it offers the possibility of posttranslational modifications. Challenges lie in the low expression rates in the nuclear genome when compared to other expression systems. Therefore the first step in this work was to test whether selected Chlorella virus PBCV-1 promoters, do lead to enhanced expression rates, but there was no detectable expression. Furthermore synthetic repeats of heat shock elements were used in combination with the endogenous RBCS2-promoter and analysed for expression rates via reporter measurements. The combination of synthetic heat shock elements in eightfold repeats in combination with RBCS2 enhanced expression rates of luciferase after heat shock up to threefold in comparison with the up to now strongest known promoter combination HSP70A-RBCS2. Basalexpression turned out to be best for the HSE1x-RBCS2 promoter and reached expression levels fivefold higher compared to HSP70A-RBCS2. To examine if the artificial heat shock elements (HSEs) are bound by the heat shock factor 1 in C. reinhardtii a ChIP assay with HSE8x-RBCS2 and the antibody of HSF1 of C. reinhardtii was done. It could be shown that HSF1 binds HSEs and therefore one can explain the heat shock inducibility of HSEs is regulated via the conserved heat shock response. In contrast to luciferase fluorescence reporters do not need substrate. Because of this advantage and the possibility of FACS analyses and fluorescence microscopy tagRFP was established as new reporter in C.reinhardtii. Enhancement of expression rates through new constitutive and inducible promoter combinations and the possible use of tagRFP as new fluorescence reporter are significant steps in establishing C.reinhardtii as expression system for products in biotechnology.

Submitted by Mariana Dornelles Vargas (marianadv) on 2015-03-24T15:04:38Z No. of bitstreams: 1 alem_midias.pdf: 1130568 bytes, checksum: 20270da2e2fa0248ab2ec0d00fc8e6e4 (MD5) / Made available in DSpace on 2015-03-24T15:04:38Z (GMT). No. of bitstreams: 1 alem_midias.pdf: 1130568 bytes, checksum: 20270da2e2fa0248ab2ec0d00fc8e6e4 (MD5) Previous issue date: 2012-03-23 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Esta dissertação de mestrado tem como tema principal as práticas de repórteres internacionais, conceito que compreende os profissionais das diferentes categorias do jornalismo internacional como, por exemplo, os correspondentes internacionais, os enviados especiais e os correspondentes de guerra. O mapeamento das práticas desses jornalistas foi concentrado em estudos já publicados sobre essas profissões do jornalismo e em livros com as memórias e as experiências de repórteres em diferentes épocas, locais e cobertura de acontecimentos entre os anos de 1917 e 2011. Esses livros escritos por jornalistas se revelam como um outro espaço legítimo de produção do jornalismo, além dos meios tradicionais como os jornais, o rádio e a televisão, ao permitirem que seus próprios autores realizem a interpretação e o comentário sobre a prática jornalística. Por meio desses materiais, são trabalhadas observações que envolvem os diferentes estágios da profissão, o impacto proporcionado pelo surgimento de novas tecnologias de informação e a interferência dessa evolução junto ao conjunto das práticas e processos dos repórteres internacionais. O trabalho contempla também questões problemáticas relacionadas à idealização glamurizada da profissão, à visão crítica desses jornalistas a partir dos novos formatos do jornalismo, e, principalmente, à prática de cobertura determinada a partir do conceito de circularidade nos processos da produção de conteúdo jornalístico. Além de contribuir com os estudos que envolvem as práticas, pretende servir como elemento para compreensão dos contextos políticos e econômicos que envolvem a profissão do repórter internacional. / The main theme of this Masters Thesis is the international reporters' practices, a concept that encompasses professionals from different categories such as the international correspondents, the special envoys and the war correspondents. The mapping of these practices focused on studies already published about these branches of journalism and in books with the memories and the experiences of the reporters in different times, places and coverages between the years of 1917 and 2011. These books that were written by journalists reveal themselves as another legitimate space to produce journalism beyond the traditional means like newspapers, radio and television when they allow their own authors to perform the interpretation and comment about the practice. Through these materials, we work on observations that involve the different phases of the profession, the impact derived from the rise of new information technologies and the interference of this evolution in the set of practices and processes of the international reporters. The thesis also contemplates problematic issues related to the glamorous idealization of the profession, to the critical view of these journalists based on new formats of journalism and, mainly, the practice of coverage determined from the concept of circularity in the processes of production of journalistic content. Besides contributing to the studies that involve the practice, it intends to serve as an element of understanding of the political and economical scenarios that involve the profession of international reporter.

Submitted by Vanessa Nunes (vnunes@unisinos.br) on 2015-03-26T12:20:33Z No. of bitstreams: 1 MariaChaise.pdf: 2048554 bytes, checksum: 4852bd639d7af51f3da09f670438d994 (MD5) / Made available in DSpace on 2015-03-26T12:20:33Z (GMT). No. of bitstreams: 1 MariaChaise.pdf: 2048554 bytes, checksum: 4852bd639d7af51f3da09f670438d994 (MD5) Previous issue date: 2010 / Nenhuma / As tecnologias de informação e comunicação têm motivado a emergência de novas relações entre os sujeitos e as mídias. A internet, e em especial a web 2.0, deram a largada para a liberdade de emissão e o intercâmbio de conteúdos. Qualquer indivíduo com acesso à rede ganhou condições de produzir e disseminar informações, a qualquer momento e em qualquer lugar. Este cenário, que possui aporte significativo na crescente mobilidade das tecnologias de registro de conteúdos, confere novo status aos leitores colaboradores. Contudo, em webjornais ditos de referência, as possibilidades para que leitores sejam incorporados ao processo de produção jornalístico são pré-definidas e limitadas. Ao mesmo tempo em que convidam o público à participação, estes veículos seguem detentores do protocolo de sentido dos enunciados. Por meio de processos de seleção e mediação, definem pela inclusão ou não dos conteúdos, mesmo nos canais denominados participativos ou colaborativos. A presente pesquisa investigou as colaborações dos interagentes no canal de webjornalismo participativo Leitor-Repórter, do webjornal de referência zerohora.com. Por meio de uma análise de conteúdo, pretendeu-se compreender, de um lado, as motivações dos leitores ao enviarem materiais colaborativos à seção e, por outro lado, avaliar o processo de seleção ou mediação de conteúdo efetivado pelos profissionais editores do canal. As lógicas que regem o comportamento dos atores envolvidos nesta negociação são analisadas tendo em vista sua intersecção com os códigos tanto do jornalismo convencional quanto do jornalismo olaborativo. Além de traçar um perfil das contribuições dos leitores no canal colaborativo, o estudo também investigou o processo que é desencadeado no interior do webjornal para que se tenha uma colaboração publicada. Os resultados demonstram que o webjornalismo participativo praticado por meio da seção Leitor-Repórter atingiu com limites o status de ferramenta de democratização e, em suma, representa um canal adicional de recepção de informações, muitas vezes subaproveitadas pela redação do webjornal. / The information and communication technologies have motivated the urgency for new relationships between the subjects and the media. The Internet, especially the web 2.0, introduced the issue freedom and the exchange of subjects. Any individuals with access to the net got conditions to produce and spread information, at any given time and place. This scenario, which has a meaningful contribution within the increasing mobility of subjects record technologies, gives a new status to the collaborators / readers. However, on web newspapers, the possibilities for readers to be incorporated to the process of journalistic production are pre-defined and limited. At the same time they invite the audience to participate, these media follow the holders of sense protocol of the enunciations. By the means of selection and mediation processes, they decide to include the contents or not, even in participating or collaborative channels. This research investigated the interagents' collaborations in the online participating journalism Reader-Reporter, from reference web newspaper zerohora.com. Through a subject analysis, it was aimed to understand, on one side, the readers' motivations to send collaborative materials to the section and, on the other side, to assess the selection or mediation of subjects process carried out by the channel editing professionals. The logics that rule the behavior of the actors that are involved in this negotiation are analyzed based on their intersection with the codes of the conventional or collaborative journalism. Besides tracing a profile of the readers' contributions in the collaborative channel, the study also investigated the process that is triggered within the web newspaper in order to have a collaboration published. The outcomes show that the participative online journalism practiced through the Reader-Reporter section has achieved with limits the status of democratization tool and, in short, represents an additional channel for information reception, mostly underestimated by the web newspaper editorial staff.

Submitted by JOSIANE SANTOS DE OLIVEIRA (josianeso) on 2017-12-05T13:20:52Z No. of bitstreams: 1 Leonardo Oliveira da Silva_.pdf: 3228671 bytes, checksum: 4081098e9cf08a23833e60a09efde639 (MD5) / Made available in DSpace on 2017-12-05T13:20:52Z (GMT). No. of bitstreams: 1 Leonardo Oliveira da Silva_.pdf: 3228671 bytes, checksum: 4081098e9cf08a23833e60a09efde639 (MD5) Previous issue date: 2017-03-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / PROEX - Programa de Excelência Acadêmica / Esta pesquisa trata das novas relações entre os clubes de futebol e os veículos tradicionais de comunicação a partir dos processos de midiatização que permearam a sociedade. Parte-se do pressuposto de que, favorecidos pelos os novos dispositivos de comunicação e pelo domínio das lógicas de mídia, os clubes de futebol constituíram seus departamentos de imprensa e passaram eles próprios a produzir seus conteúdos. Deixaram de ser apenas fontes e viraram integrantes ativos do tecido comunicacional. Essa mudança fez com que alterassem a forma de conviver com os veículos tradicionais, deflagrando uma disputa entre o campo midiático e o campo esportivo. Como conceito de campo, utilizamos o formulado por Bourdieu (2004), segundo o qual se tratam de espaços autônomos, com leis próprias e integrantes de um macrocosmo. Acreditamos que essa passagem dos clubes de futebol, em relação à mídia, de uma postura reativa para posição ativa provocou o que Fausto Neto (2008) definiu como zonas de afetação entre setores de uma sociedade midiatizada. Entendemos, assim como José Luiz Braga (2012), que estão nesses novos modos de se relacionar dessa sociedade o alvo para o qual devemos direcionar nossas pesquisas. Para estudar esse novo modelo de relacionamento entre clubes de futebol e repórteres setoristas e as afetações que causam na produção jornalística, nos debruçamos sobre os casos do Sport Club Internacional e do jornal Zero Hora, ambos de Porto Alegre, usando a análise de conteúdo como principal metodologia de estudo, conjugada com pesquisa exploratória e entrevistas. A partir de observação do material publicado pelo site oficial do clube e pelo ColoradoZH, entendemos que as alterações nas relações entre fonte e repórter estão mudando a cobertura esportiva nos veículos tradicionais de comunicação e colocando no cenário midiático um novo integrante, a mídia oficial dos clubes de futebol. / This research deals with the new relationship between football clubs and traditional media outlets, after the mainstream media processes have been a part of society. You start with the assumption that, favoured by the new communication devices and by knowing how to handle this new kind of media, football clubs have assembled their press offices and started, themselves, to produce new content. The clubs have stopped being only sources of information and became active members of the communicational world. That change made the whole relationship with mainstream media different, which created a dispute bewtween the media field and the sports field. By field, we use the concept formulated by Bordieu (2004), in which he claims they're autonomous spaces, with their own laws, both integrating a macrocosmos. We believe that this shift in football clubs and their relation to the media, from being a reactive to an active agent, provoked what Fausto Neto (2008) has defined as "zones of affectation" among sectors of a mediated society. We understand, as José Luiz Braga (2012) does, that the targets in which we must point our researches are in this new relations with society. To study this new relationship way involving football clubs, specialized reporters and the affectations that they cause in the journalistic process, we looked onto the cases of Sport Club Internacional and the newspaper Zero Hora, both from Porto Alegre, using content analysis as the main study methodology, along with exploratory research and interviews. From observing the material published at the club's official website and at ColoradoZH, we understand that the alterations in the relations between source and reporter are shifting the sports coverage at the traditional media outlets, and puting at the media scene a new member: the offical media of the football clubs.

Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.

Esta dissertação tem como objetivo analisar em que medida o telejornal Repórter Brasil Noite, exibido na emissora pública TV Brasil, pratica um jornalismo que contribui com o direito à informação, discutido a partir da reflexão teórica sobre a construção da cidadania e a sua interface com o jornalismo. As conceituações clássicas da cidadania foram analisadas, fazendo um paralelo com o desenvolvimento histórico do jornalismo. Percebemos que os valores atribuídos à prática do jornalismo ao longo da sua história determinaram a sua interface com o Estado e com as demandas de direitos referentes a essa prática, num movimento que culminou no direito à informação, que pode ser considerado como direito civil, político e social. O jornalismo é compreendido como uma forma de conhecimento gerada por meio de procedimentos da prática profissional alimentado pelos acontecimentos cotidianos. No entanto, por sua configuração comercial predominante, os veículos de comunicação, reproduzem a ideologia de produtividade nas redações, o que impede a reflexão necessária na prática jornalística, e atuam deliberadamente defendendo seus interesses econômicos. Em tese, o veículo público pode dar condições para a prática do jornalismo livre das determinações econômicas inerentes aos meios privados. Para tanto, um veículo público de comunicação deve ter como base um modelo de gestão e financiamento que permita sua autonomia com relação aos governos e ao mercado. A pesquisa empírica consistiu na coleta de dados de uma amostragem de seis edições do telejornal em semana artificial, submetida a uma análise quantitativa e, numa etapa posterior, a uma análise qualitativa de uma matéria selecionada de cada um dessas seis edições. Constatamos, por fim, que a TV Brasil encontra dificuldades para produzir um jornalismo que contribua plenamente para o direito à informação. / This dissertation aims to analyze to what extent the newscast Repórter Brasil Noite, broadcasted by public television TV Brasil, practices a journalism that contributes to the right of information, concept discussed from theoretical reflexion over the citizenship development and its interface with journalism. The classic concepts of citizenship were analyzed, drawing a parallel with the journalism history. We noticed that the values attributed to journalism practice along its history had defined its interface with the state and with demands of rights regarding this activity, in a movement that culminated in the right of information, which might be considered as a civil, political and social right. Journalism is understood as a form of knowledge generated through procedures of the professional practice fed by quotidian events. However, due to the predominant commercial settings, media vehicles reproduce the ideology of productivity in the newsrooms, what restrains the reflexion needed in journalistic activity, and act deliberately defending their economic interests. Presumably, the public media vehicle might create conditions for the journalism activity free from economic determinations inherent to the commercial vehicles. Therefore, a public vehicle must be based on a management and financing model that allows its autonomy from governments and market influences. The empirical research consisted in data collection from a sample of six editions of the newscast in an artificial week submitted to a quantitative analysis and, in a posterior stage, to a qualitative analysis of one report selected from each edition. We noticed that TV Brasil experiences difficulties to fully contribute for the right of information.

Thesis (M. Environmental Science (Water Science))--North-West University, Potchefstroom Campus, 2007.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.

Overexpression of Phosphoenolpyruvate carboxykinase - cytosolic (PEPCK, encoded by Pck-1 gene) has been found to be associated with the prevalence of hyperglycemia in Type 2 Diabetes Mellitus (T2DM) patients. The Pck-1 enzyme catalyzes the rate limiting step in endogenous glucose production. The aims of this study are to develop a Pck-1:eGFP reporter zebrafish and validate it as a potential tool for the screening of novel anti-diabetic compounds. 3.6 kb zebrafish Pck-1 promoter fragment was cloned and a Pck-1:eGFP expression vector was constructed. After DNA microinjection, we generated Pck-1:eGFP reporter zebrafish with strong eGFP expression in developing liver. Validation studies confirmed that Pck-1:eGFP zebrafish embryos responded to treatment of glucose, cAMP and dexamethasone, metformin and rosiglitazone similarly to that of humans. This novel Pck-1:eGFP reporter fish line can serve as a tool for the screening and development of novel anti-diabetic drugs that may have potential in the treatment of T2DM.