Conditional Transgenesis Using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCre×R26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12–rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05–0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48–72 h, with an EC(50) of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals

Expression of the DiCre constructs in tissues of adult or embryonic transgenic animals.

<p>A: Northern blots obtained from DiCre ES cells or tissues from young adult DiCre transgenic animals (two independent samples for each), indicating the level of mRNA for the indicated transgenes. B: Expression level, relative to that measured in the liver, of the same transgenes in tissues of adult animals, as measured by qPCR (means +/− SEM, n = 3–5). C: Northern blot, and D: qPCR analysis, as in panels 7A and 7B, from embryonic (E15) tissues and adult tissues used as comparison. Note that the adult tissues used here were not the same as those shown on panels 7A and 7B.</p

Induction of Cre-mediated recombination in DiCre×Z/EG mice.

<p>A: The presence of recombination is shown by EGFP expression in the heart of DiCre×Z/EG adult animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). B: In the absence of rapamycin treatment no specific fluorescence can be seen.</p

Induction of Cre-mediated recombination in primary cultures of embryonic tissues of DiCre×R26R mice.

<p>The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product on the bright-field (upper row) and phase contrast (lower row) images of the same fields, in primary cultures prepared from various tissues of DiCre×R26R E15 embryos. Cultures were exposed between 3 and 7 day <i>in vitro</i> to 10 nM AP23102.</p

Induction of Cre-mediated recombination in various tissues of DiCre×R26R mice.

<p>The presence of recombination is shown by ß-galactosidase expression, as revealed by the blue X-Gal reaction product, in various tissues of adult DiCre×R26R animals ten days after the end of treatment with the inducer (5×10 mg/kg rapamycin i.p.). Bars represent 100 µm. The insert for the liver shows the total absence of recombination in the absence of rapamycin treatment.</p

Targeting strategy for the creation of DiCre ES cells.

<p>A: Scheme of the targeting vector and of the Rosa26 locus following homologous recombination. “B” and “R” stand, respectively, for the BamHI and EcoRV sites that are used when screening ES cells clones for correct insertions using Southern blots and the probes indicated on the scheme. Fragments amplified when genotyping animals are also represented B: Representative examples of Southern blots obtained from wild type ES cells or clones with correct recombination, using the probes shown on the schemes above.</p

Outcomes and adverse events of pre- and extensively drug-resistant tuberculosis patients in Kinshasa, Democratique Republic of the Congo: A retrospective cohort study.

BackgroundExtensively drug-resistant tuberculosis (XDR TB) is a very serious form of tuberculosis that is burdened with a heavy mortality toll, especially before the advent of new TB drugs. The Democratic Republic of the Congo (DRC) is among the countries most affected by this new epidemic.MethodsA retrospective analysis was performed of the records of all patients with pre- and extensively drug-resistant tuberculosis hospitalized from January 1, 2015 to December 31, 2017 and monitored for at least 6 months to one year after the end of their treatment in Kinshasa; an individualized therapeutic regimen with bedaquiline for 20 months was built for each patient. The adverse effects were systematically monitored.ResultsOf the 40 laboratory-confirmed patients, 32 (80%) patients started treatment, including 29 preXRB and 3 XDR TB patients. In the eligible group, 3 patients (9.4%) had HIV-TB coinfections. The therapeutic success rate was 53.2%, and the mortality rate was 46.8% (15/32); there were no relapses, failures or losses to follow-up. All coinfected HIV-TB patients died during treatment. The cumulative patient survival rate was 62.5% at 3 months, 53.1% at 6 months and 53.1% at 20 months. The most common adverse events were vomiting, Skin rash, anemia and peripheral neuropathy.ConclusionThe new anti-tuberculosis drugs are a real hope for the management of Drug Resistant tuberculosis patient and other new therapeutic combinations may improve favorable outcomes

A reservoir of brown adipocyte progenitors in human skeletal muscle

Brown adipose tissue uncoupling protein-1 (UCP1) plays a major role in the control of energy balance in rodents. It has long been thought, however, that there is no physiologically relevant UCP1 expression in adult humans. In this study we show, using an original approach consisting of sorting cells from various tissues and differentiating them in an adipogenic medium, that a stationary population of skeletal muscle cells expressing the CD34 surface protein can differentiate in vitro into genuine brown adipocytes with a high level of UCP1 expression and uncoupled respiration. These cells can be expanded in culture, and their UCP1 mRNA expression is strongly increased by cell-permeating cAMP derivatives and a peroxisome-proliferator-activated receptor-{gamma} (PPAR{gamma}) agonist. Furthermore, UCP1 mRNA was detected in the skeletal muscle of adult humans, and its expression was increased in vivo by PPAR{gamma} agonist treatment. All the studies concerning UCP1 expression in adult humans have until now been focused on the white adipose tissue. Here we show for the first time the existence in human skeletal muscle and the prospective isolation of progenitor cells with a high potential for UCP1 expression. The discovery of this reservoir generates a new hope of treating obesity by acting on energy dissipation. <br /