Variable Expression of Cre Recombinase Transgenes Precludes Reliable Prediction of Tissue-Specific Gene Disruption by Tail-Biopsy Genotyping

The Cre/loxP-system has become the system of choice for the generation of conditional so-called knockout mouse strains, i.e. the tissue-specific disruption of expression of a certain target gene. We here report the loss of expression of Cre recombinase in a transgenic mouse strain with increasing number of generations. This eventually led to the complete abrogation of gene expression of the inserted Cre cDNA while still being detectable at the genomic level. Conversely, loss of Cre expression caused an incomplete or even complete lack of disruption for the protein under investigation. As Cre expression in the tissue of interest in most cases cannot be addressed in vivo during the course of a study, our findings implicate the possibility that individual tail-biopsy genotypes may not necessarily indicate the presence or absence of gene disruption. This indicates that sustained post hoc analyses in regards to efficacy of disruption for every single study group member may be required

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Determinants of Contraceptive Availability at Medical Facilities in the Department of Veterans Affairs

OBJECTIVE: To describe the variation in provision of hormonal and intrauterine contraception among Veterans Affairs (VA) facilities. DESIGN: Key informant, cross-sectional survey of 166 VA medical facilities. Data from public use data sets and VA administrative databases were linked to facility data to further characterize their contextual environments. PARTICIPANTS: All VA hospital-based and affiliated community-based outpatient clinics delivering services to at least 400 unique women during fiscal year 2000. MEASUREMENTS: Onsite availability of hormonal contraceptive prescription and intrauterine device (IUD) placement. RESULTS: Ninety-seven percent of facilities offered onsite prescription and management of hormonal contraception whereas 63% offered placement of IUDs. After adjusting for facility caseload of reproductive-aged women, 3 organizational factors were independently associated with onsite IUD placement: (1) onsite gynecologist (adjusted odds ratio [OR], 20.35; 95% confidence interval [CI], 7.02 to 58.74; P<.001); (2) hospital-based in contrast to community-based practice (adjusted OR, 5.49; 95% CI, 1.16 to 26.10; P=.03); and (3) availability of a clinician providing women's health training to other clinicians (adjusted OR, 3.40; 95% CI 1.19 to 9.76; P=.02). CONCLUSIONS: VA's provision of hormonal and intrauterine contraception is in accordance with community standards, although onsite availability is not universal. Although contraception is a crucial component of a woman's health maintenance, her ability to obtain certain contraceptives from the facility where she obtains her primary care is largely influenced by the availability of a gynecologist. Further research is needed to determine how fragmentation of women's care into reproductive and nonreproductive services impacts access to contraception and the incidence of unintended pregnancy