The sgRNA pool of 3 guide RNAs targeting FERMT2 and the scramble control were obtained from Synthego. The sgRNA pellets were rehydrated in 1X TE buffer to make a stock of 100 μM. A working solution of 30 μM sgRNA was made fresh before electroporating the cells. For every reaction, the Ribonucleoprotein complex was assembled by adding 3 μl of30 μM sgRNA to 0.5 μl of 20 μM Cas9 at a ratio of 9:1 in 3.5 μl resuspension buffer R and incubated for 10 min at room temperature.
Electroporation of LNCaP cells was achieved by an implemented electroporation device system according to manufacturer’s instructions . The Neon Transfection System 10 μL kit was used for the transfection of human prostate cancer cells. Cells were cultured 48 h before electroporation and harvested at nearly 80% confluency. Cells at a density of 2 × 10were washed with PBS and resuspended in 5 μl resuspension buffer R .
. As a control, PC3 cells were transfected with a non-targeting scrambled control shRNA . DU145 cells were transfected with pCMV6-Entry vector carrying RTN4RL2 or with the empty vector as a control . Cells were plated in a 6-well plate and grown overnight at 37 °C. The next day, cells were washed with PBS and incubated with 1 mL serum-free media at 37 °C for 2 h. For transfection, 4 μg of plasmid was mixed with 12 μL of lipofectamine 2000 in 200 μL of serum-free RPMI media.
Source: Healthcare Press (healthcarepress.net)
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Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium - BMC BiologyBackground Methods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we developed an efficient method achieving this long-sought goal. Results We used ultrasound-guided microinjections to transduce the embryonic endoderm at day 8 (E8.0) in utero. The injection procedure can be completed in 20 min and had a 100% survival rate. By injecting a small volume (0.1–0.2 μl) of concentrated virus, single shRNA constructs as well as lentiviral libraries can successfully be transduced. The new method stably and reproducibly targets adult intestinal epithelium, as well as other endoderm-derived organs such as the lungs, pancreas, liver, stomach, and bladder. Postnatal analysis of young adult mice indicates that single transduced cells at E8.0 gave rise to crypt fields that were comprised of 20–30 neighbouring crypts per crypt-field at 90 days after birth. Lentiviral targeting of ApcMin/+ mutant and wildtype mice revealed that heterozygous loss of Apc function suppresses the developmental normal growth pattern of intestinal crypt fields. This suppression of crypt field sizes did not involve a reduction of the crypt number per field, indicating that heterozygous Apc loss impaired the growth of individual crypts within the fields. Lentiviral-mediated shRNA knockdown of p53 led to an approximately 20% increase of individual crypts per field in both Apc+/+ and ApcMin/+ mice, associating with an increase in crypt size in ApcMin/+ mice but a slight reduction in crypt size in Apc+/+ mice. Overall, p53 knockdown rescued the reduction in crypt field size in Apc-mutant mice but had no effect on crypt field size in wildtype mice. Conclusions This study develops a novel technique enabling robust and reproducible in vivo targeting of intestinal stem cells in situ in the unperturb
Source: medical_xpress - 🏆 101. / 51 Read more »
Norwich City pay tribute to eight-year-old fan who diedPlayers and staff at Norwich City honour Amber Sheehy, who died from cancer last weekend. Rest in Peace.😢
Source: BBCNews - 🏆 3. / 97 Read more »
King Charles 'to make Prince Edward the Duke of Edinburgh after change of heart'The King had initially favourited Prince Charlotte for the prestigious role, according to reports. Should the people of Edinburgh not have a say in this ? monarchy is so outdated 🤷♂️ Tell this goon we dont want him No one cares
Source: Daily_Record - 🏆 9. / 89 Read more »
Transient Receptor Potential Ankyrin-1-expressing vagus nerve fibers mediate IL-1β induced hypothermia and reflex anti-inflammatory responses - Molecular MedicineBackground Inflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1β (IL-1β) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines. Methods To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1β. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1β-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant. Results Here, we describe a previously unrecognized mechanism by which IL-1β activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis. Conclusion Thus, IL-1β activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inf
Source: BioMedCentral - 🏆 22. / 71 Read more »