Download Blue Htv 3.1 Beta 3
Download Blue Htv 3.1 Beta 3 ->>> https://fancli.com/2t8lcJ
Scatterplot depicting the correlation of (A) Shannon and (B) Simpson with the number of hours after initial treatment with live or heat-treated viruses. The alpha diversity indices are shown on the y axis and the hours after initial treatment are depicted on the x-axis. The black line denotes the linear regression line with the gray shading indicating a 95% confidence interval. Spearman correlation indices and P-values are shown in the upper left corner of each panel. Points are colored by vessel type (D5-HTV, blue; D5-LV orange; D25A-HTV purple; D25A-LV green).
Scatterplot depicting the correlation between Bray-Curtis distance to time zero (T0) and the number of hours after initial treatment with live or heat-treated viruses. The Bray-Curtis distance to T0 is shown on the y axis and the hours after initial treatment are depicted on the x-axis. Color lines denote the linear regression lines for each vessel with the shading indicating a 95% confidence interval. Spearman correlation indices and P values are shown in the upper left corner. Points and lines are colored by vessel type (D5-HTV, blue; D5-LV orange; D25A-HTV purple; D25A-LV green).
Scatterplot depicting the correlation between the relative abundance of the dominant bacterial phylum and the number of hours after initial treatment with live or heat-treated viruses. The relative abundance of phylum Bacteroidetes, Firmicutes, Verrucomicrobia, Proteobacteria, and Actinobacteria are shown on the y-axis, and the hours after initial treatment are depicted on the x-axis. The black line denotes the linear regression line with the gray shading indicating a 95% confidence interval. Spearman correlation indices and P values are shown in the corner of each panel. Points are colored by vessel type (D5-HTV, blue; D5-LV orange; D25A-HTV purple; D25A-LV green).
Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.
In this study, we developed an improved version of PA-Cre called PA-Cre 3.0, which is based on the same blue-light-dependent dimerization system, Magnets. We demonstrate the improved efficiency of PA-Cre 3.0 and its applications in vivo using newly generated mouse lines expressing PA-Cre 3.0 conditionally. We believe this improved system and mouse model availability can enhance genetic studies in living systems to address biological hypotheses and unveil the molecular and pathophysiological mechanisms underlying various diseases.
To validate the function of PA-Cre 3.0 in vivo, we induced transient expressions of the CAG promoter-driven Magnets-based constructs, both PA-Cre 3.0 and the original one, using HTV injection into mice. Following the plasmid DNA injection, mouse abdomens were exposed to blue light to induce recombination, following the experimental setup of our previous study in which the Luc reporter plasmids were also used with the PA-Cre construct10. However, the validation of Cre-loxP recombination for targeting a single pair of loxP sites is essential for applying the PA-Cre system in vivo. To address this, we utilized Ai14:Floxed-tdTomato heterozygous reporter mouse line, which can express tdTomato red fluorescent proteins in a Cre-loxP recombination-dependent manner18. The original PA-Cre showed higher leakiness in the dark and ambient light conditions whereas the codon-optimized version PA-Cre 3.0 had no spontaneous Cre-loxP recombination at all in the mouse livers (Fig. 2d and Supplementary Fig. 5). Also, we found that the mice injected with PA-Cre 3.0 showed higher recombination efficiency and tdTomato reporter expressions than the original PA-Cre. These results reveal that PA-Cre 3.0 is applicable for blue-light-dependent Cre-loxP recombination in the single loxP pair target in mouse model in vivo. Also, this result suggests that bioluminescence quantification in live mouse livers using Luc reporter10 might not be as reliable and appropriate as tdTomato fluorescent reporter in individual liver cells in order to detect such leak activity of Cre-loxP recombination in vivo tissues.
To apply PA-Cre 3.0 to mammalian primary cells, we transduced this construct into mouse primary neural progenitor cells isolated from Ai14: Floxed-tdTomato heterozygous reporter mouse brains. To express PA-Cre 3.0, we applied adeno-associated virus (AAV) using the neuronal activity-dependent promoter, RAM element, and a chemical-controllable system, Tet-off19 (Fig. 4a). Under blue-light conditions with phorbol 12-myristate 13-acetate (PMA), a PKC activator, or high KCl stimuli as depolarization induction, we observed successful Cre-loxP recombination mediated by AAV-RAM-Tet-off-PA-Cre 3.0 in Ai14 mouse-derived neurons (Supplementary Fig. 7a, b). In addition, we confirmed that doxycycline addition prevented light-dependent Cre-loxP recombination via the Tet-off system in vitro. As additional experiments, we tested a doxycycline-fed condition and light stimulation of mouse brain amygdala, which responds to stress, and confirmed that the Tet-off system was functional in the AAV-based mouse model in vivo (Supplementary Fig. 7c) and is consistent with the in vitro results in the primary neural cells (Supplementary Fig. 7a, b). Furthermore, we applied the AAV-RAM-Tet-off-PA-Cre 3.0 viruses into the mouse brain dorsal raphe nucleus, which also responds stress, together with mCherry reporter viruses, and fibrotic light sources. Under blue-light conditions following AAV infection, we observed Cre-lox recombination in c-fos-positive neurons in the mouse brain region while there was no Cre-loxP recombination observed in mice kept in dark (Fig. 4b, c). These results reveal that AAV-RAM-Tet-off-PA-Cre 3.0 virus is a useful resource applicable for mouse brains in vivo.
To better facilitate in vivo studies, we generated a new mouse line by inserting PA-Cre 3.0 construct in the Rosa26 locus (Rosa26-CAG-Frt-Stop-Frt-PA-Cre 3.0, B4 mouse embryonic stem cell clone, Fig. 5a). This mouse line enables for tissue-specific expression of PA-Cre 3.0, following Flp-Frt recombination. To confirm that this targeting strategy is functional in mice, we isolated mouse embryonic fibroblasts (MEFs) from embryos of PA-Cre 3.0 B4 mouse line crossed with Ai14: Floxed-tdTomato reporter mice. We observed the expression of tdTomato red fluorescent proteins, following Flp lentiviral infection and blue-light exposure, revealing the successful Cre-loxP recombination without unintended leakiness in the dark condition (Fig. 5b). As an infection control, the same lentiviral system expressing yellow fluorescent proteins was tested in the MEFs (Supplementary Fig. 8). In addition, we could observe strong and specific tdTomato expressions in livers of PA-Cre 3.0 B4: Ai14 mice, following HTV injections of CAG promoter-mediated Flp plasmids into the mice (Fig. 5c and Supplementary Fig. 9).
Because mouse breeding takes time for experimental design with multiple allele uses, an all-in-one version containing both PA-Cre 3.0 and a fluorescent protein reporter will be valuable for users. Also, transient expression of PA-Cre 3.0 might be ideal as Cre recombination is transient and irreversible and continuous Cre activity might induce neuronal toxicity20. To accomplish this, we added loxP sites in the front and back of PA-Cre 3.0 construct as a self-deficient option in the Rosa26 locus wherein the PA-Cre 3.0 can be removed by itself, following blue-light stimulation leaving only the mKate2 red fluorescent protein reporter expression (Fig. 6a). The advantage of this targeting design is an all-in-one concept for PA-Cre 3.0 and reporter expressions in the single construct. To validate this system, MEFs were isolated from the all-in-one version of PA-Cre 3.0 mouse line (clone A20) crossed with Rosa26-FLPe line. We confirmed that blue-light illumination could induce mKate2 expression, revealing that illumination induces the successful recombination by PA-Cre 3.0 and there is no spontaneous recombination in dark at all (Fig. 6b, c). The primary cortical neurons isolated from the all-in-one PA-Cre 3.0 A20 mouse line and infected with Flp lentiviruses also showed mKate2 expression with blue-light illumination (Fig. 6d, e). The results demonstrate that the all-in-one PA-Cre 3.0 A20 mouse line is a useful and reliable resource in mouse genetic study.
The HTC Vive headset worked on macOS 10.14 (Mojave) and gave smooth rendering similar to Windows 10 using a Radeon Vega 56 external GPU. Setting up the eGPU to run on Mac laptops older than 2017 where it is not officially supported by Apple poses problems. A specific beta version of SteamVR is needed called macos_default, and there were no special setup problems. The Vive Pro headset also works with eGPU and in addition works with an iMac with Radeon Pro 580 or 575 graphics and no eGPU.
The current SteamVR beta macos_default is version 1539100633, built Oct 9, 2018 at 08:57. This is a year old with no updates and no longer works with the Valve labeled basestation 2.0 (see Sept 30 comment below). It would not be surprising if Mac SteamVR breaks in the next 6 months due to a Steam update or macOS update. Since Valve is not maintaining SteamVR on macOS that will be the end of VR on Mac. Since 99% of VR use is on Windows, it is best to use Windows if possible. 2b1af7f3a8