Temporally-Controlled Site-Specific Recombination in Zebrafish

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia

Measurement of associated W plus charm production in pp collisions at √s=7 TeV

Peer reviewe

Ligand-dependent Cre-mediated recombination.

<p>(a) Scheme of the ligand-dependent recombination event in cells of the red-to-green reporter line. The chimeric CreER^T2 recombinase is retained in the cytoplasm in the absence of the ligand. After administration of TAM which is converted to the active ligand 4-OHT, CreER^T2 translocates to the nucleus, where it catalyzes the recombination event. (b) Expression of CreER^T2 in the diencephalon of the <i>Tg(pax2a:CreER^T2)^#19</i> line at early segmentation stages revealed by <i>in situ</i> hybridization. (c) EGFP expression in the diencephalon of double transgenic embryos at 24 hpf bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#19</i> alleles after TAM treatment at mid-gastrulation stages. (d) Expression of CreER^T2 in rhombomere 3 and 5 of the <i>Tg(pax2a:CreER^T2)^#45</i> line at early segmentation stages revealed by <i>in situ</i> hybridization. (e) EGFP expression in rhombomere 3 and 5 of double transgenic embryos at 24 hpf bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#45</i> alleles after TAM treatment at mid-gastrulation stages. Abbreviations: f, forebrain; h, hindbrain; m, midbrain. Scale bar, 50 µm.</p

Kinetics of ligand-dependent Cre-mediated recombination.

<p>(a) Expression of CreER^T2 in the <i>Tg(pax2a:CreER^T2)^#19</i> line at the 12-somite stage revealed by <i>in situ</i> hybridization. (b) Control embryos treated with DMSO never show any EGFP. (c) Immunofluorescence staining with antibodies to EGFP is detectable 4 hours after application of TAM and expanded further after 6 hours. (d) Onset of EGFP expression by immunofluorescence staining is detected after 2 hours and expanded further after 4 and 6 hours after application of 4-OHT. a–d Dorsal views of double transgenic embryos at 12-, 16-, 20 and 24-somite stage (15, 17, 19 and 21 hpf). Abbreviations: f, forebrain; e, eye anlage; h, hours; m, midbrain. Scale bar, 30 µm.</p

Cre-mediated recombination in the red-to-green reporter line.

<p>(a) Scheme of the recombination event. In the absence of Cre the <i>EF1</i>α promoter drives the expression of DsRed2 but changes to EGFP after successful Cre-mediated recombination. (b) Embryos of the red-to-green reporter line show strong DsRed2 and no EGFP fluorescence. (c) Maternal contribution of the <i>Tg(hsp70:EGFP-Cre)</i> allele results in complete loss of DsRed2 and ubiquitous EGFP expression in double transgenic embryos. (d) Paternal contribution of the <i>Tg(hsp70:EGFP-Cre)</i> allele leads to strong DsRed2 and mosaic EGFP expression in double transgenic embryos. (e) Paternal contribution of the <i>Tg(hsp70:EGFP-Cre)</i> allele and brief heat induction at mid-gastrulation stages results in reduced DsRed2 and strong ubiquitous EGFP expression in double transgenic embryos. (f) Embryos of the <i>Tg(hsp70:EGFP-Cre)</i> line show only weak EGFP fluorescence after brief heat induction at mid-gastrulation stages. b–f Lateral views of live 24 hpf embryos bearing different transgenes. Scale bar, 125 µm.</p

Dose-dependent recombination in the <i>Tg(pax2a:CreER^T2)^#19</i> and <i>Tg(pax2a:CreER^T2)^#45</i> lines by TAM.

<p>(a) Double transgenic embryos bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#19</i> alleles show strong EGFP expression in the diencephalon after application of 5 µM TAM at mid-gastrulation stages. Application of 0.5 µM TAM or 0.05 µM TAM at the same stage, results in reduced EGFP expression or single EGFP-positive cells, respectively. (b) Double transgenic embryos bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#45</i> alleles show strong EGFP expression in rhombomere 3 and 5 after application of 5 µM TAM at mid-gastrulation stages. Application of 0.5 µM TAM at the same stage, results in single EGFP-positive cells. a, b Dorsal views of double transgenic embryos at 24 hpf. Scale bar, 30 µm.</p