Journal: Cell Reports Methods

Article Title: A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity

doi: 10.1016/j.crmeth.2023.100673

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney 293 (HEK293) cells were cultured in DMEM high glucose supplemented with 10% FBS and 1% P-S. HEK293 cells stably expressing a nuclear localized Cre recombinase (SC004-Puro, GenTarget Inc.) were cultured in DMEM high glucose supplemented with 10% FBS, 1% P-S, 2 mM L-glutamine (Gibco), and non-essential amino acids (Gibco) as suggested by the vendor.

Techniques: Recombinant, Electron Microscopy, Ligation, Sequencing, Expressing, Software, Imaging

Journal: Nature Communications

Article Title: VCAM1 acts in parallel with CD69 and is required for the initiation of oligodendrocyte myelination

doi: 10.1038/ncomms13478

Figure Lengend Snippet: ( a ) Tissue lysates from 7-, 21- and 28-day-old CD69 shRNA transgenic (Tg) and control mouse brains were immunoblotted with an antibody against MBP, CNPase, CD69, VCAM1 or actin. Data are representative of three experiments. ( b ) Corpus callosum sections at postnatal day 11 were co-stained with an anti-MBP antibody (red) and DAPI (blue). Data are representative of two experiments. Scale bar, 200 μm. ( c – e ) Electron microscopic data of 9-day-old corpus callosum are shown. Data are representative. Scale bar, 2 μm. The g-ratios (200–300 myelinated fibres) are plotted for axon diameters. Average g-ratios are also shown. Data were evaluated using Student’s t -test (** P =7.13E−15; n =213–260 axons of two independent experiments).

Article Snippet: MBP promoter-driven Cre recombinase transgenic mice (RBRC No. 01461) were obtained from Riken BioResource Center (Ibaraki, Japan).

Techniques: shRNA, Transgenic Assay, Staining

Journal: Science signaling

Article Title: Fluorescent Ca 2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions

doi: 10.1126/scisignal.aal2039

Figure Lengend Snippet: (a) Representative images of astrocytes loaded with Rhod-2 AM (4.5 μM) or prepared from GFAPCre:GCaMP3fl transgenic mice (GFAP-GCaMP3). Scale bar, 100 μm. (b) GCaMP3 expression in astrocytes has no effect on Na,K-ATPase-mediated 86Rb+ uptake. GFAP-GCaMP3 astrocytes showed no difference in ouabain-sensitive 86Rb+ uptake. Expression of GCaMP3 induced by adenovirus bearing Cre recombinase (100 MOI, 6 days post-transduction) in cultured astrocytes prepared from GCaMP3fl transgenic mice also had no effect on ouabain-sensitive 86Rb+ uptake (AdV-GCaMP3). Conversely, Rhod-2 AM (4 μM) loading decreased ouabain-sensitive 86Rb+ uptake in all groups of cultured mouse astrocytes. n=6–23 wells; *p<0.05, **p<0.01, ****p<0.0001 compared to (−) Rhod-2 AM of control group (0.1% DMSO). (c) Spontaneous Ca2+ activity in cultured mouse astrocytes. Number of spontaneous Ca2+ events were detected by Fluo-4 AM (2 and 4 μM), Rhod-2 AM (2.25 and 4.5 μM) or GCaMP3. In groups combining Ca2+ imaging of GCaMP3 with Rhod-2 AM, number of Ca2+ events detected with Rhod-2 (left, orange channel) and GCaMP3 (right, green channel) were both counted. n=4–6 wells; ****p<0.0001 compared to GFAP-GCaMP3 control group (0.1% DMSO). (d) Histogram comparing number of spontaneous Ca2+ events in Adv-GCaMP3 expressing mouse astrocytes in the presence and absence of 5,5’-Dibromo BAPTA (p=0.4188, unpaired t test n=4–8 wells). Displayed are means ± S.E.M.

Article Snippet: Titration of adenoviral infection of GCaMP3fl astrocytes To induce GCaMP3 expression in cortical mouse astrocyte cultures prepared from GCaMP3fl mice, cells were infected with adenovirus bearing Cre recombinase under the control of the constitutive cytomegalovirus (CMV) promoter (courtesy of Aris N. Economides from Regeneron Pharmaceuticals, Tarrytown, NY) for 5 h with 10, 50 or 100 multiplicities of infection (MOI).

Techniques: Transgenic Assay, Expressing, Transduction, Cell Culture, Control, Activity Assay, Imaging