53 research outputs found
Comparison between C57BL/6N and C57BL/6J for their systolic blood pressure and heart rate responses to various salt challenges measured by NIBP.
<p>Measurements were made during the dark periods of a modified light/dark cycle. NS = normal salt diet (n = 20 and n = 20, for C57BL/6N and C57BL/6J respectively), LS = low salt diet (n = 20 and n = 20, for C57BL/6N and C57BL/6J respectively), HS = high Na<sup>+</sup>/normal K<sup>+</sup> diet (n = 9 and n = 10, for C57BL/6N and C57BL/6J respectively) and HS/LK = high Na<sup>+</sup>/low K<sup>+</sup> diet (n = 9 and n = 10, for C57BL/6N and C57BL/6J respectively). One-way ANOVA per light phase followed by Tukey Kramer’s post-hoc test; #: p<0.05 HS/LK compared to HS.</p
Effects of high-salt/normal potassium and high-salt/low potassium on mean blood pressure and heart rate in C57BL/6N mice.
<p>Mice were monitored with telemetry in the dark (A, B, C) and light (C) periods of a modified light/dark cycle. NS = normal salt diet (n = 17), LS = low salt diet (n = 15), HS = high Na<sup>+</sup>/normal K<sup>+</sup> diet (n = 8) and HS/LK = high Na<sup>+</sup>/low K<sup>+</sup> diet (n = 6). One-way ANOVA per light phase followed by Tukey’s post-hoc test; *: p<0.05 compared to NS diet; #: p<0.05 HS/LK compared to HS.</p
Effect of high salt diets on the circadian blood pressure variations in C57BL/6N and C57BL/6J mice under a reverse light/dark cycle.
<p>2.5 days continuous telemetric recordings of systolic blood pressure after 2 weeks of NS, HS or HS/LK diet challenge. A) n = 8 per group B) n = 6 per group C) n = 8 per group D) n = 9 per group. Two-way ANOVA followed by Sidak’s post-hoc test *: p<0.05 for interaction.</p
Sodium and potassium levels in urine and plasma in C57BL/6N.
<p>Sodium and potassium levels in urine and plasma in C57BL/6N.</p
Comparison between C57BL/6N and C57BL/6J for their mean blood pressure and heart rate responses to various salt challenges.
<p>Mice were monitored with telemetry during the dark (A, B) and light (C) periods of a modified light/dark cycle. NS = normal salt diet (n = 17 and n = 17, for C57BL/6N and C57BL/6J respectively), LS = low salt diet (n = 15 and n = 17, for C57BL/6N and C57BL/6J respectively), HS = high Na<sup>+</sup>/normal K<sup>+</sup> diet (n = 8 and n = 8, for C57BL/6N and C57BL/6J respectively) and HS/LK = high Na<sup>+</sup>/low K<sup>+</sup> diet (n = 6 and n = 9, for C57BL/6N and C57BL/6J respectively). One-way ANOVA per light phase followed by Tukey’s post-hoc test; *: p<0.05 compared to NS diet; #: p<0.05 HS/LK compared to HS.</p
Plasma ion, aldosterone and renin activity measurements in C57BL/6N and C57BL/6J male mice.
<p>Plasma ion, aldosterone and renin activity measurements in C57BL/6N and C57BL/6J male mice.</p
Effects of various salt challenges on mean blood pressure and heart rate in C57BL/6N male mice.
<p>Mice were monitored with telemetry and placed in a standard (A, C) or modified light/dark cycle (B, D). NS = normal salt diet (n = 12 and n = 17, for the standard and modified light cycle respectively), LS = low salt diet (n = 11 and n = 15, for the standard and modified light cycle respectively), HS = high Na<sup>+</sup>/normal K<sup>+</sup> diet (n = 5 and n = 6, for the standard and modified light cycle respectively). One-way ANOVA per light phase followed by Tukey’s post-hoc test; *: p<0.05 compared to NS diet.</p
Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.
BackgroundRecent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders.Methodology/principal findingsIn order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles.ConclusionTargeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis
RF313, an orally bioavailable neuropeptide FF receptor antagonist, opposes effects of RF-amide-related peptide-3 and opioid-induced hyperalgesia in rodents
Although opiates represent the most effective analgesics, their use in chronic treatments is associated with numerous side effects including the development of pain hypersensitivity and analgesic tolerance. We recently identified a novel orally active neuropeptide FF (NPFF) receptor antagonist, RF313, which efficiently prevents the development of fentanyl-induced hyperalgesia in rats. In this study, we investigated the properties of this compound into more details. We show that RF313 exhibited a pronounced selectivity for NPFF receptors, antagonist activity at NPFF1 receptor (NPFF1R) subtype both in vitro and in vivo and no major side effects when administered in mice up to 30 mg/kg. When co-administered with opiates in rats and mice, it improved their analgesic efficacy and prevented the development of long lasting opioid-induced hyperalgesia. Moreover, and in marked contrast with the dipeptidic NPFF receptor antagonist RF9, RF313 displayed negligible affinity and no agonist activity (up to 100 μM) toward the kisspeptin receptor. Finally, in male hamster, RF313 had no effect when administered alone but fully blocked the increase in LH induced by RFRP-3, while RF9 per se induced a significant increase in LH levels which is consistent with its ability to activate kisspeptin receptors. Altogether, our data indicate that RF313 represents an interesting compound for the development of therapeutic tools aiming at improving analgesic action of opiates and reducing adverse side effects associated with their chronic administration. Moreover, its lack of agonist activity at the kisspeptin receptor indicates that RF313 might be considered a better pharmacological tool, when compared to RF9, to examine the regulatory roles of RF-amide-related peptides and NPFF1R in reproduction
Analysis of the G1 animals interrogated by ONT sequencing for the <i>Pam</i>-flox project.
NB. No Sanger sequencing was performed on the Pam floxed G1 generation prior to analysis with ONT due to project timelines. The figure shows the PCR amplification of the genomic region of interest with (A) Pam-F1 and Pam-R1 primers (1431 bp amplicon) and (B) LoxPF and LoxPR primers (801 bp amplicon) from biopsies taken from Pam-3’s offspring. (C) The table details the first litter obtained by mating Pam-flox-3 with a WT mouse. The ID, outcome of PCR amplification of the regions of interest as well as the initial conclusion for each individual are shown. Sanger data obtained subsequent to the ONT run is displayed in (D) and (E) for animals Pam-3.1a and Pam-3.1b respectively. (D) The panels show the sequencing of PCR amplicon obtained from animal Pam-3.1a with Pam-F1 and Pam-R1. NHEJ events at each intended loxP insertion site are highlighted in blue, demonstrating that the LoxP product was generated by an off-target integration of the donor. (E) The panels show the sequencing of PCR amplicon obtained from animal Pam-3.1b with Pam-F1 and Pam-R1. LoxP insertion sites are highlighted in blue. However, when sequencing the LoxP PCR amplicons, more than one trace is present indicating multiple LoxP alleles and rearrangements. Animal(s) interrogated by ONT sequence analysis are highlighted in green. + is positive control amplified from an unrelated (A) WT, (B) floxed animal. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (TIF)</p
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