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

Stratum continuum of Information: Scolarly communications and the role of university libraries

Abstract As a consequence of digitization universities have to investigate their scholarly communication process. In fact, this is a quest for values that goes beyond the issues of the day. Once found, these values operate as criteria for assessing competencies, roles and instruments. The communication process must allow for free exchange of ideas and results. In particular, knowledge created in the public domain must be open accessible. The process must be reliable, lend credibility to authors and give support to users. The respective competencies are logistics, validation and mediation. New technologies do not only support these classical aspirations, but develop their own dynamics when it comes to broadening the communication scope. Publications can be enhanced by including audio-visuals, executable files and datasets. Linking techniques enable the creation of organically growing bodies of knowledge. Who is the fittest for this scholarly communication job? Is it universities and their libraries and university presses, or could it best remain in the hands of global publishing monopolies?Delft University of Technolog

On-line vs DC-ROM delivery of information

Considers the use of new technologies in library information delivery systems. Discusses the virtues of paper, CD-ROM, and online media in terms of accessibility, timeliness, compactness, searchability, preservability, authenticity, and appearance, as well as end user benefit. Surmises that information is a means to an end to the end user, who wants the easy access and speed offered by paper media, with the language and help availability benefits of electronic media.Delft University of Technolog

The library as a hybrid organisation

In the last decade technology exploded into libraries. It impacted not only library work processes but imported also new strategic options. Library systems, i.e. databases, imported notions of project management and efficiency and resulted in catalog sharing. The Internet with Gopher and Veronica, brought co-operation in the field of document supply and collection co-ordination. The most consequential technology, however, is the Web. It combines instant publishing, hyperlinking, interactivity and multimediality. And it's so easy to apply. Numerous new actors will make their entry into the information chain which, of course, means competion. Libraries need to define their position in this Webbed World. They have to enter the market place at least partially which transforms them into hybrid organizations, both vanguard and debatable.TU Delft LibraryDelft University of Technolog

Towards a New System of Scholarly Communications

TU Delft LibraryDelft University of Technolog