Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

17-β-Estradiol-dependent regulation of somatostatin receptor subtype expression in the 7315b prolactin secreting rat pituitary tumor in vitro and in vivo

In the present study, we have investigated the role of estrogens in the regulation of somatostatin receptor subtype (sst) expression in 7315b PRL- secreting rat pituitary tumor cells in vitro and in vivo. sst were undetectable in freshly dispersed cells of the transplantable 7315b tumor. When 7315b cells were cultured in medium containing 10% FCS, the number of high affinity sst increased with prolonged culture time. However, when the medium was supplemented with 10% horse serum (HS) instead of FCS, no sst were detectable on 7315b cells even after three weeks of culturing. In contrast to HS, FCS contains high E2-levels (HS, 8 pM; FCS, 134 pM). The antiestrogen tamoxifen (0.5 μM) significantly inhibited the sst number to 50.5% of the value of untreated FCS-grown cells, suggesting that E2 stimulates sst expression in 7315b rat pituitary tumor cells. E2 (l0 nM) induced a rapid increase in sst number in HS-grown 7315b cells. Octreotide (1μM) significantly inhibited PRL release and the intracellular PRL concentration of 7315b cells that were cultured in medium supplemented with FCS or with HS + l0 nM E2 but not in HS alone. This indicates that the sst present on these cells are biologically active. RT-PCR analysis revealed that none of the five currently known sst subtypes were present in freshly dispersed 7315b pituitary tumor cells. The expression of sst2- and sst3- messenger RNA (mRNA) was unequivocally correlated to the presence of E2 because these sst subtypes were detected only in cells that were cultured for7 and 14 days in medium supplemented with FCS or with HS + 10 nM E2. sst1, sst4 and sst5 messenger RNA could not be detected. The 7315b tumor itself synthesizes and secretes huge amounts of PRL. The high PRL levels in tumor-bearing rats inhibit the ovarian E2-production. No detectable E2 levels could be measured in the serum of 7315b tumor-bearing rats. The sc administration of 20 μg/day E2-benzoate normalized the circulating E2 levels in 7315b tumor- bearing rats. Moreover, E2-treatment indeed induced sst expression in vivo as shown by ligand binding studies using membrane homogenates and [125I- Tyr3]-octreotide as radioligand and by autoradiography on tissue sections. In agreement with the in vitro studies, the expression of the sst2 subtype was established by RT-PCR analysis in 7315b tumors of E2-treated rats. However, in contrast to the in vitro studies. E2-treatment did not effectuate the expression of the sst3 subtype, suggesting that the in vitro stimulus of E2 is stronger. In conclusion: 1) sst2 and sst3 expression in the 7315b rat prolactinoma model is primarily dependent upon the presence of estrogens; 2) the antihormonal action of octreotide in 7315b tumor cells in vitro is mediated via the sst2 and/or sst3 subtypes; 3) the absence of sst expression in vivo can be explained by the hormonal environment of the 7315b tumor cells. The 7315b tumor cells in vivo may down-regulate their own receptor status via their host, because of the ensuing hyperprolactinemia results in a hypo-estrogenic state.</p

Improved science-based transformation pathways for the development of safe and sustainable plastics

Environmental Biolog

Protein-protein interactions and genome engineering : novel strategies to study cell polarity

Cell polarity is a fundamental property of cells. The identification of conserved polarity regulators that control polarity in a variety of distinct tissues raises a number of questions. How are the same components used and integrated in tissue-specific ways to give rise to the wide variety of polarized tissues? What are the downstream components through which the polarity program acts? We developed a tissue-specific affinity purification/mass spectrometry approach for C. elegans based on in vivo biotinylation of Avi-tagged proteins of interest by the bacterial biotin ligase BirA. Tissue-specific biotinylation is accomplished by expressing BirA from a tissue-specific promoter, while the Avi-tagged protein is expressed from its native regulatory sequences. Biotinylated bait proteins are subsequently purified and interacting proteins identified by mass spectrometry. We confirmed the tissue-specificity of our approach and applied it to several polarity proteins. Tissue-specific purification of DLG-1 and CDC-42 resulted in the identification of several known interaction partners, demonstrating that our approach can identify valid interactions from specific tissues. It is important to gain a detailed mechanistic understanding of the proteins that regulate polarity. The functioning of a protein can in large part be dictated by the protein interactions it engages in. Here, we expand the concept of Y2H-based interaction domain mapping to the genome-wide scale. We generated a human prey library by fragmenting an ORFeome collection with ultrasonication and demonstrated the quality of the library by screening it with polarity and cell division proteins. We identified several interactions previously described in literature as well as novel interactions, and validated 55% of all identified interactions by affinity purifications in cell culture. The best approach for studying the function of a protein in vivo is to precisely modify the genome to express a mutated protein that for example lacks interaction domains. We developed the CRISPR/Cas9 system for C. elegans to be able to specifically engineer its genome. By expressing a guide RNA the Cas9 endonuclease is targeted to a specific locus in the genome where it creates a double strand break. Imprecise repair of the break can yield mutations and we obtained mutants for all four genes tested. Several other groups independently adapted the CRISPR/Cas9 system for C. elegans, and we reviewed the different approaches taken. We used our CRISPR/Cas9 approach to gain a better understanding of the functioning of the Crumbs proteins in C. elegans. While the Crumbs protein is essential for epithelial polarity and viability in Drosophila, the two Crumbs homologs identified in C. elegans thus far do not appear to play a major role in polarity establishment. We identified a third Crumbs homolog in C. elegans, which localizes in a polarized pattern in several tissues. We developed a variation of our CRISPR/Cas9 approach to delete entire genes, and generated a triple Crumbs deletion mutant. Remarkably, animals lacking all three Crumbs homologs are viable. Overexpression of all three Crumbs homologs caused changes in the localization pattern of the polarity protein PAR-3, suggesting that the C. elegans Crumbs homologs play a non-essential role in polarity establishment

Microplastics in binnenlucht

De laatste jaren is er veel aandacht voor hele kleine plastic deeltjes, microplastics, in het milieu. Ze breken heel langzaam of niet af en worden overal in het milieu gevonden. Er is nog niet zoveel bekend over microplastics in de lucht in huis (binnenlucht). Het ministerie van Infrastructuur en Waterstaat (IenW) wil daar graag meer over weten. Van sommige stoffen zijn kleine deeltjes namelijk niet goed voor de luchtkwaliteit, en daarmee voor de volksgezondheid. Het RIVM heeft daarom verkend of microplastics in de binnenlucht voorkomen. Het heeft hiervoor een overzicht gemaakt van de kennis in de wetenschappelijke literatuur over microplastics binnenshuis. Met die kennis kan IenW zo nodig maatregelen nemen. De informatie kan ook worden gebruikt om te beoordelen of er risico’s voor de gezondheid zijn en welke gegevens daarvoor nodig zijn. Over het algemeen lijken de concentraties laag te zijn. Gemiddeld zijn het er tussen de 1,6 en 9,3 microplastic deeltjes per kubieke meter. De gemeten deeltjes zijn vrij groot, groter dan 11 micrometer. Ze zijn daarmee groter dan de veel kleinere deeltjes fijnstof die standaard voor de luchtkwaliteit worden gemeten (PM2.5 en PM10, oftewel kleiner dan 2,5 en 10 micrometer). Het is mogelijk dat er ook veel kleinere microplastics in de onderzochte binnenlucht zitten. Maar het is nu nog moeilijk om de kleinere microplastics in de lucht te meten. Juist de kleinere deeltjes zijn niet goed voor de luchtkwaliteit (PM2.5 en PM10). De belangrijkste bronnen van microplastics in huis zijn textiel, zoals kleding, tapijt en gordijnen. Naast vezels worden veel fragmenten van microplastics in binnenlucht gevonden. Uitgezocht moet worden of deze deeltjes ook van de vezels van textiel komen of van andere bronnen. Daarnaast is informatie nodig over aanwezigheid van de kleinste microplastic deeltjes. Met deze kennis kunnen effectievere maatregelen worden genomen om de luchtkwaliteit te verbeteren.In recent years, the problem of tiny plastic particles, or microplastics, in the environment has attracted considerable attention. Microplastics degrade slowly, if at all, and are found everywhere around us. Not much is known about microplastics in air inside homes. The Ministry of Infrastructure and Water Management (IenW) would very much like to know more about this because small particles of certain substances are detrimental to air quality and consequently to public health. RIVM has therefore looked into the possible presence of microplastics in indoor air and has summarised the knowledge found in scientific literature on this topic. This knowledge will enable IenW to take any measures that may be necessary. It can also be used to assess whether these substances entail health risks and the data needed to make such assessments. Generally speaking, the concentrations appear to be low. On average, there are between 1.6 and 9.3 microplastic particles per cubic metre. The particles measured are fairly large, i.e. more than 11 micrometres in diameter. This makes them larger than the ultrafine particles normally measured to assess air quality (PM2.5 and PM10 or smaller than 2.5 and 10 micrometres in diameter). It is possible that much smaller microplastics are also present in the investigated homes. But it is currently difficult to measure the smaller types of microplastic particles in air. It is precisely smaller particles that are not good for air quality (e.g. PM2.5 and PM10). The most significant sources of microplastics in homes are textiles, such as clothing, carpets and curtains. Besides fibres, many fragments of microplastics are found in indoor air. Further research is needed to determine whether these particles also originate from textile fibres or from other sources. Besides this, information is needed on the smallest type of microplastic particles. With this knowledge more effective measures can be taken to improve air quality.Ministerie van I&