As células da granulosa (CG) são constituintes do ambiente folicular de suma importância para o desenvolvimento, maturação e aquisição da competência oocitária, desempenhando funções como a esteroidogênese, expressão de receptores de LH (LHR) e síntese de inúmeras proteínas essenciais. Contudo, a funcionalidade e ação destas células são dependentes de alguns fatores derivados do oócito, como o GDF9 e o BMP15. A ação desses fatores, por sua vez, depende da sinalização via receptor BMPRII, já identificado em células da granulosa de mamíferos, porém com suas funções ainda pouco conhecidas na espécie bovina. O silenciamento gênico por lipofecção consiste num método de transfecção eficiente e pouco agressivo para as células, representando uma importante ferramenta para o estudo funcional de genes e proteínas celulares. O objetivo do presente trabalho foi, primeiramente, otimizar as condições de lipofecção em CG bovinas e, a partir desse protocolo, estabelecer uma metodologia de silenciamento gênico para o BMPRII usando a técnica de RNA de interferência, a fim de possibilitar a futura utilização de tal estratégia como ferramenta para o estudo funcional do BMPRII e outros genes de interesse. Para tanto, no primeiro experimento referente à otimização das condições de lipofecção em CG bovinas, onde as células da granulosa, obtidas de ovários oriundos de abatedouro foram tratadas com diferentes quantidades dos lipofectores Lipofectamine® RNAiMAX (1, 2 e 3µl) ou Lipofectamine™ 2000 (1, 2 e 3µl) e dos indicadores de transfecção siGLO® (30, 50, 75 e 100nM) ou plasmídeo transgênico FUGW (100, 200, 300, 400, 600 e 900 nM) durante 24 e 48 horas de cultivo. A melhor eficiência de lipofecção foi observada às 24 horas de cultivo com 2µl de Lipofectamine™ 2000 + 100nM de siGLO®. Num segundo experimento, a partir das condições de lipofecção determinadas, foram testadas diferentes concentrações do siBMPRII (0 a 500 pmol) durante 24 h de cultivo. Ao final desse período, as células de cada grupo foram avaliadas quanto à abundância relativa mRNA BMPRII por PCR em tempo real. Todas as concentrações resultaram em uma redução semelhante de transcritos, sendo possível adotar a menor concentração (100 pM), a qual foi utilizada para determinar o tempo mínimo de cultivo necessário para obter eficiência no processo de silenciamento. As células foram tratadas por 0, 6, 12, 18 e 24 h e também avaliadas quanto aos transcritos de BMPRII. Os tempos de incubação que proporcionaram maior redução do mRNA para o BMPRII foram 18 e 24 horas. A melhor concentração e tempo de incubação com o siRNA foram avaliados por Western Blotting, confirmando a redução da expressão de BMPRII também em nível de proteína. Em conclusão, este trabalho possibilitou o estabelecimento de uma metodologia eficiente para o silenciamento gênico por lipofecção do BMPRII em CG bovinas, a qual poderá ser utilizada como ferramenta para o estudo funcional de diversos genes de interesse. / Granulosa cells (GC) are part of the folicular environment and are important for the development, maturation and acquisition of developmental competence of oocytes, performing functions such as steriodogenesis, expression of LH receptors (LHR) and synthesis of several important proteins. The function of these cells is dependent on some oocyte-derived factors, such as GDF9 and BMP15. Signaling of these factors involves the activation of the BMPRII receptor identified in granulosa cells, however, its function is not well established in bovine cells. Gene silencing is an important tool for functional studies of different cellular proteins. The aim of the present study was to develop a protocol for gene silencing using the RNA interference technique by lipofecction to knockdown BMPRII expression, for future functional studies on the role of this receptor and other genes of interest in bovine granulosa cells. For this purpose, the first experimente aimed to optimize lipofection conditions in bovine granulosa cells, collected from abbattoir ovaries and cultured in vitro. Lipofecction was tested for different concentrations of two diferent lipofection agents (1, 2 and 3 µl Lipofectamine® RNAiMAX or Lipofectamine™ 2000) and two transfection indicators (30, 50, 75 and 100nM siGLO® or 100, 200, 300, 400, 600 and 900 nM transgenic plsamid FUGW) for 24 and 48 h in culture. Best lipofection efficiency was observed at 24 h culture with 2µl Lipofectamine™ 2000 + 100nM siGLO®, which was used for the next experiment. The second experiment tested different BMPRII-siRNA concentrations (0 to 500 pM) for 24 h. At the end of culture the cells were be assessed for the relative abundance for BMPRII by real time PCR. All concentrations similarly reduced transcript abundance relative to control (0 pM). For the third experiment, the lowest effective concentration was used (100 pM) to test different culture periods with the BMPRII-siRNA and determine the minimum period to obtain transcript reduction. Cells will be treated for 0, 6, 12, 18 and 24 h and assessed for BMPRII transcripts. Greater reduction in BMPRII transcripts was observed after 18 and 24 h. The best concentration (100 pM) and time (24 h) were confirmed for knockdown of the BMPRII protein by western blotting. In conclusion, this work has provided the establishment of a reliable method for gene silencing using lipofection for the BMPRII in bovine granulosa cells, which may be used for functional studies for this gene and others of interest.

As células da granulosa (CG) são constituintes do ambiente folicular de suma importância para o desenvolvimento, maturação e aquisição da competência oocitária, desempenhando funções como a esteroidogênese, expressão de receptores de LH (LHR) e síntese de inúmeras proteínas essenciais. Contudo, a funcionalidade e ação destas células são dependentes de alguns fatores derivados do oócito, como o GDF9 e o BMP15. A ação desses fatores, por sua vez, depende da sinalização via receptor BMPRII, já identificado em células da granulosa de mamíferos, porém com suas funções ainda pouco conhecidas na espécie bovina. O silenciamento gênico por lipofecção consiste num método de transfecção eficiente e pouco agressivo para as células, representando uma importante ferramenta para o estudo funcional de genes e proteínas celulares. O objetivo do presente trabalho foi, primeiramente, otimizar as condições de lipofecção em CG bovinas e, a partir desse protocolo, estabelecer uma metodologia de silenciamento gênico para o BMPRII usando a técnica de RNA de interferência, a fim de possibilitar a futura utilização de tal estratégia como ferramenta para o estudo funcional do BMPRII e outros genes de interesse. Para tanto, no primeiro experimento referente à otimização das condições de lipofecção em CG bovinas, onde as células da granulosa, obtidas de ovários oriundos de abatedouro foram tratadas com diferentes quantidades dos lipofectores Lipofectamine® RNAiMAX (1, 2 e 3µl) ou Lipofectamine™ 2000 (1, 2 e 3µl) e dos indicadores de transfecção siGLO® (30, 50, 75 e 100nM) ou plasmídeo transgênico FUGW (100, 200, 300, 400, 600 e 900 nM) durante 24 e 48 horas de cultivo. A melhor eficiência de lipofecção foi observada às 24 horas de cultivo com 2µl de Lipofectamine™ 2000 + 100nM de siGLO®. Num segundo experimento, a partir das condições de lipofecção determinadas, foram testadas diferentes concentrações do siBMPRII (0 a 500 pmol) durante 24 h de cultivo. Ao final desse período, as células de cada grupo foram avaliadas quanto à abundância relativa mRNA BMPRII por PCR em tempo real. Todas as concentrações resultaram em uma redução semelhante de transcritos, sendo possível adotar a menor concentração (100 pM), a qual foi utilizada para determinar o tempo mínimo de cultivo necessário para obter eficiência no processo de silenciamento. As células foram tratadas por 0, 6, 12, 18 e 24 h e também avaliadas quanto aos transcritos de BMPRII. Os tempos de incubação que proporcionaram maior redução do mRNA para o BMPRII foram 18 e 24 horas. A melhor concentração e tempo de incubação com o siRNA foram avaliados por Western Blotting, confirmando a redução da expressão de BMPRII também em nível de proteína. Em conclusão, este trabalho possibilitou o estabelecimento de uma metodologia eficiente para o silenciamento gênico por lipofecção do BMPRII em CG bovinas, a qual poderá ser utilizada como ferramenta para o estudo funcional de diversos genes de interesse. / Granulosa cells (GC) are part of the folicular environment and are important for the development, maturation and acquisition of developmental competence of oocytes, performing functions such as steriodogenesis, expression of LH receptors (LHR) and synthesis of several important proteins. The function of these cells is dependent on some oocyte-derived factors, such as GDF9 and BMP15. Signaling of these factors involves the activation of the BMPRII receptor identified in granulosa cells, however, its function is not well established in bovine cells. Gene silencing is an important tool for functional studies of different cellular proteins. The aim of the present study was to develop a protocol for gene silencing using the RNA interference technique by lipofecction to knockdown BMPRII expression, for future functional studies on the role of this receptor and other genes of interest in bovine granulosa cells. For this purpose, the first experimente aimed to optimize lipofection conditions in bovine granulosa cells, collected from abbattoir ovaries and cultured in vitro. Lipofecction was tested for different concentrations of two diferent lipofection agents (1, 2 and 3 µl Lipofectamine® RNAiMAX or Lipofectamine™ 2000) and two transfection indicators (30, 50, 75 and 100nM siGLO® or 100, 200, 300, 400, 600 and 900 nM transgenic plsamid FUGW) for 24 and 48 h in culture. Best lipofection efficiency was observed at 24 h culture with 2µl Lipofectamine™ 2000 + 100nM siGLO®, which was used for the next experiment. The second experiment tested different BMPRII-siRNA concentrations (0 to 500 pM) for 24 h. At the end of culture the cells were be assessed for the relative abundance for BMPRII by real time PCR. All concentrations similarly reduced transcript abundance relative to control (0 pM). For the third experiment, the lowest effective concentration was used (100 pM) to test different culture periods with the BMPRII-siRNA and determine the minimum period to obtain transcript reduction. Cells will be treated for 0, 6, 12, 18 and 24 h and assessed for BMPRII transcripts. Greater reduction in BMPRII transcripts was observed after 18 and 24 h. The best concentration (100 pM) and time (24 h) were confirmed for knockdown of the BMPRII protein by western blotting. In conclusion, this work has provided the establishment of a reliable method for gene silencing using lipofection for the BMPRII in bovine granulosa cells, which may be used for functional studies for this gene and others of interest.

Septins are evolutionarily conserved proteins with roles in chromosome congression and segregation, cytokinesis and microtubule destabilization. Septins form homo- and hetero-oligomeric complexes, which are thought to act as dynamic scaffolds. We identified SEPT2/9/11/10 as novel interacting partners of adenomatous polyposis coli (APC), a bona fide tumor suppressor. Since septins and APC have similar roles and knockdown phenotypes, I sought to determine if they work together to perform their cellular functions. I showed that APC co-immunoprecipitates with endogenous septins in colon cancer cell lines. Using siRNA, I found that SEPT2 and APC may function within the same pathway to regulate DNA congression and segregation. Co-depleting SEPT9 with APC slightly alleviates the chromosome congression and segregation defects caused by siAPC alone. siSEPT9 increased abscission times, which was rescued by co-depleting APC. Future studies should elucidate the significance of the rescue data obtained upon APC and SEPT9 co-depletion and APC’s interactions with SEPT10/11.

Septins are evolutionarily conserved proteins with roles in chromosome congression and segregation, cytokinesis and microtubule destabilization. Septins form homo- and hetero-oligomeric complexes, which are thought to act as dynamic scaffolds. We identified SEPT2/9/11/10 as novel interacting partners of adenomatous polyposis coli (APC), a bona fide tumor suppressor. Since septins and APC have similar roles and knockdown phenotypes, I sought to determine if they work together to perform their cellular functions. I showed that APC co-immunoprecipitates with endogenous septins in colon cancer cell lines. Using siRNA, I found that SEPT2 and APC may function within the same pathway to regulate DNA congression and segregation. Co-depleting SEPT9 with APC slightly alleviates the chromosome congression and segregation defects caused by siAPC alone. siSEPT9 increased abscission times, which was rescued by co-depleting APC. Future studies should elucidate the significance of the rescue data obtained upon APC and SEPT9 co-depletion and APC’s interactions with SEPT10/11.

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of EGFR is a common feature in variety of human cancers including lung, colorectal, breast, pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi) pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated through the transfection of three specifically designed short hairpin RNAs (shRNAs) against human EGFR. The shRNAs were specifically designed using bioinformatics tools and their individual knockdown efficacy determined through the introduction of an exogeneous based target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show substantial potential for the specific and efficient targeting of EGFR in human cancer cells.

No description available.

In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.

Le système génétique complexe des mitochondries de plantes supérieures n'a pu être étudié par des approches transgéniques car les méthodes conventionnelles ne permettent pas de transformer ces organites. Une approche alternative a été développée au laboratoire, grâce à l'existence d'un processus naturel assurant l'import d'ARN de transfert (ARNt) du cytosol dans les mitochondries. Il a été montré qu'un mime d'ARNt peut servir in vivo de navette pour importer dans les mitochondries de plante des ARN-passagers exprimés à partir de transgènes nucléaires. L'utilisation d'un transribozyme comme séquence-passagère a permis d'obtenir l'invalidation spécifique d'un ARN messager (ARNm) majeur dans les mitochondries de cellules végétales transformées. Nous avons mis en oeuvre cette stratégie pour développer des études de régulation mitochondriale. Cinq ARNm mitochondriaux (nad9, sdh3, cob, cox3 et atp9) ont été choisis comme cibles pour des transribozymes spécifiques à tête de marteau. Après validation de l'activité de ces ribozymes in vitro, les vecteurs d'expression portant les transgènes correspondants ont servi pour transformer des cultures cellulaires de Nicotiana tabacum, des plantes d'Arabidopsis thaliana (pour nad9, cob, cox3 et atp9) et des plantes de N. tabacum (pour sdh3). L'invalidation spécifique des ARN mitochondriaux ciblés par les ribozymes a été établie in vivo. La réponse, en termes de régulation, à l'invalidation des cibles individuelles a été analysée au niveau de l'ensemble du transcriptome. Alors qu'il a été généralement considéré jusqu'à présent que les processus de régulation mitochondriaux chez les plantes se passent essentiellement au stade post-transcriptionnel, nos résultats sont fortement en faveur de mécanismes de coordination des ARNm dans les mitochondries et entre les organites et le noyau. / The complex genetic system of higher plant mitochondria could not be studied by transgenic approaches because conventional methods do not permit genetic transformation of these organelles. An alternative approach has been developed in the laboratory, thanks to the existence of a natural process of transfer RNA (tRNA) import from the cytosol into mitochondria. It was shown that a tRNA mimic can be used in vivo as a shuttle for importing into plant mitochondria passenger RNAsexpressed from nuclear transgenes. Taking a trans-cleaving ribozyme as a passenger sequence allowed to obtain the specific knockdown of a major messenger RNA (mRNA) in the mitochondria of transformed plant cells. We used this strategy to develop mitochondrial regulation studies. Five mitochondrial mRNAs (nad9, sdh3, cob, cox3 and atp9) were chosen as targets for specific transcleaving hammerhead ribozymes. After validating the in vitro activity of these ribozymes, the corresponding expression constructs served to transform Nicotiana tabacum cells, Arabidopsis thaliana plants (for nad9, cob, cox3 and atp9) and N. tabacum plants (for sdh3). Specific in vivo ribozyme-mediated knockdown of the targeted mitochondrial RNAs was established. The regulation response to the knockdown of the individual targets was analyzed at the whole transcriptome level.Whereas it has been generally considered so far that mitochondrial regulation processes in plants essentially occur at the post-transcriptional stage, our results strongly support mRNA coordination mechanisms within the organelles and between the organelles and the nucleus.

We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-FectTM (vehicle), and delivered as repeated daily bolus doses (0.5 mug to 4 mug) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-FectTM complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.

Primordial germ cells (PGCs) are the precursors of the germ cell lineage that eventually differentiate into mature spermatozoa and oocytes. Although present throughout the animal kingdom, the specification and migration of PGCs differs widely between species. In vertebrates, avians are evolutionary divergent from mammals and therefore allow a comparative system in which to study germ cell development in higher organisms. Unlike mouse, PGCs can be isolated from the chicken embryo, expanded and cultured long term in vitro. Analysis of these cells showed that cultured chicken PGCs maintain the characteristics of their in vivo counterparts, including the expression of key germ cell specific markers and cell surface adhesion proteins, and thus, are an ideal system to study germ cell biology. Further characterisation revealed that an avian homologue of the zinc finger transcription factor PRDM14, essential for the specification of the mammalian germ cell lineage, was expressed in chicken PGCs. cPRDM14 was found to be expressed in PGCs in vitro and in vivo from early developmental stages until expression is lost by embryonic day 10 and subsequently re-expressed in the adult testis. The expression of cPRDM14 suggested that this gene may play a conserved role in the avian germ cell lineage. To investigate the function of cPRDM14, a novel single piggyBac transposon vector containing a reverse tetracycline activator protein and a tetracycline response element-regulated promoter was developed. Testing of the integrated transposon revealed that expression was tightly regulated and it was possible to conditionally express one gene product whilst simultaneously reducing the expression of a second gene, both in vitro and in vivo. This vector system was fully functional in PGCs, and was used to create transgenic founder chickens capable of having gene expression manipulated in germ cells at various developmental stages. Transgenic offspring were produced and the transgene was inducible at early developmental stages in the G1 animals. The un-induced transgene proved to be toxic to early embryos so a transgenic line of birds could not be produced. The inducible transposon was used to knockdown cPRDM14 expression in chicken PGCs. Knockdown of this gene led to reduced PGC numbers and increased cell death, both in vitro and in ovo. Expression of the pluripotency factor cNANOG was also significantly reduced which may explain the increased cell death. The knockdown of cPRDM14 also led to an increased susceptibility of PGCs to spontaneously de-differentiate to pluripotent embryonic germ cells (EGCs). cPRDM14 knockdown PGCs exhibited elevated levels of phosphorylated ERK, a target of the FGF signalling pathway. It was possible to prevent de-differentiation of the knockdown PGCs by removing ectopic FGF from the media. Furthermore, a sustained high level of FGF signalling in the media was sufficient to drive the de-differentiation of control PGCs to EGCs, suggesting that increased FGF signalling was key to the de-differentiation process. Extensive epigenetic remodelling of mouse PGCs occurs during embryonic development and PRDM14 was shown to be involved in this process. Chicken PGCs in vitro, contain several key histone modifications (H3K4me3, H3K9me2 and H3K27me3) and are 5-methyl cytosine (5-mC) positive. Immunohistochemical analysis of these markers in PGCs, at various stages during early embryonic development, suggests that these cells do not undergo the extensive epigenetic remodelling found in their mammalian counterparts. In contrast to the mouse germ cell lineage, knockdown of cPRDM14 in cultured PGCs had no noticeable effect on the epigenetic status of chicken PGCs. Together these results demonstrate that cPRDM14 is essential for the survival and maintenance of germ cell identity in chicken PGCs, but may not be critical for maintaining the epigenetic status of these cells.