Journal: Nature Communications

Article Title: Fibroblastic reticular cells in lymph node potentiate white adipose tissue beiging through neuro-immune crosstalk in male mice

doi: 10.1038/s41467-023-36737-0

Figure Lengend Snippet: a–d Sham-operated and iLNX male C57BL/6 N mice were exposed to 6 °C or 30 °C for 2 days. a , b ELISA analysis of protein level (a) and the mRNA expression of IL-33 ( b ) in scWAT of sham-operated and iLNX mice ( n = 5). c The mRNA level of IL-33 in scWAT and iLN ( n = 8). d IL-33 protein level in iLN determined by Western blot (top) and densitometric quantification of IL-33 (bottom) ( n = 3). e – n Lentivirus encoding FLAG-tagged Cre and luciferase (Lenti-Ccl19-Cre) or luciferase only (Lenti-Ccl19-Luci) driven by the Ccl19 promoter was directly injected into iLNs (7.5 × 10 6 Transduction Units [TU] per side) of eight-week-old male IL33 fl/fl mice. Seven days after lentiviral injection, mice were housed at 30 °C for 3 weeks followed by 2-day cold exposure (6 °C) or continued to be housed at 30 °C for another 2 days. e IVIS Lumina imaging analysis showing luminescence intensity in mice with (Lenti-Ccl19-Cre) or without lentivirus injection (negative control) after intraperitoneal injection of luciferin (150 mg/kg) for 10 min. f Immunofluorescent staining with primary antibodies against Cre recombinase or gp38 (a cell surface marker of FRCs) in iLN section; Scale bar, 50 μm g The mRNA level of IL-33 in iLN ( n = 5). h ELISA analysis for IL-33 protein level in scWAT ( n = 5). i , j Quantification of IL-5, IL-13 ( i ) and MetENK ( j ) in ILC2s using flow cytometric analysis ( n = 5). k , l Flow cytometric analysis of absolute numbers of eosinophils ( k ) and M2 macrophages ( l ) ( n = 5). m The mRNA expression of thermogenic genes in scWAT, determined by real-time PCR ( n = 5). n Western blot analysis for UCP1 protein expression in scWAT (top) and densitometric quantification of UCP1 (bottom) ( n = 3). All samples are biologically independent replicates. Data are presented as mean ± SEM. Statistical data were assessed using unpaired two-tailed Student’s test ( c , d, n ) or Mann–Whitney U test (a , g – m) . All the p values were two-sided. Source data are available as a Source Data file. kDa, relative molecular weight in kilodalton. See also Fig. , – .

Article Snippet: Subsequently, cells were fixed and permeabilized before staining with rabbit mAb anti-Cre recombinase (1:400, Cell Signaling Technology, D3U7F) followed by a secondary goat anti-rabbit Alexa Fluor® 568 antibody (1:800, Thermo Fisher Scientific, #A11011).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Luciferase, Injection, Transduction, Imaging, Negative Control, Staining, Marker, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY, Molecular Weight

Journal: bioRxiv

Article Title: STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

doi: 10.1101/2021.12.08.471715

Figure Lengend Snippet: (A) Schematic of procedure for excising the positive selection cassettes and vector backbone following integration of the donor vector into the Bxb1-LP. Dashed lines indicate the sequences excised. Half arrows indicate primer binding sites with dotted lines representing the resulting PCR amplicons. (B) PCR screening using primer pairs indicated in (A), confirming the reduction in amplicon length upon expression of Cre recombinase (+). (C) Quantification of integrated hiPSCs that have excised the auxiliary sequences following Cre expression. n=5 independent transfections; error bars represent ± SEM.

Article Snippet: hiPSCs were transfected by lipofection with either an Flp-expressing plasmid (1.6 μg, pCAG_FlpoIRESpuro ( )), a Cre-expressing plasmid (1.6 μg, pEFBOS_CreIRESpuro ( )) or StemMACSTM Cre Recombinase mRNA (200 ng, Miltenyi Biotec).

Techniques: Selection, Plasmid Preparation, Binding Assay, Amplification, Expressing, Transfection

Journal: bioRxiv

Article Title: STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

doi: 10.1101/2021.12.08.471715

Figure Lengend Snippet: (A) Schematic of STRAIGHT-IN procedure to perform targeted heterozygous modifications to a 50.6 kb genomic region on chromosome 7 that includes KCNH2 . Half arrows indicate primer binding sites with dotted lines representing the resulting PCR-generated amplicons. Dashed lines indicate the sequences excised by Cre recombinase. (B) PCR products amplified with the corresponding primer pairs indicated in (A), confirming targeting of Bxb1-LP to KCNH2 ( KCNH2 +/Acc ), and subsequent re-introduction of wildtype KCNH2 ( KCNH2 +/Acc-wt ). Auxiliary sequences detected upon integration (+) were excised following Cre expression (-). The sizes of the amplicons are indicated. (C) ddPCR confirming that KCNH2 +/Acc and KCNH2 +/Acc-wt hiPSCs contained the expected number of copies of the genomic genes KCNH2 (1 and 2 copies respectively), NOS3 and AOC1 (both 2 copies), and the Bxb1-LP cassette transgenes, eGFP and BsdR (1 and 0 copies respectively). Error bars represent Poisson 95% CI. (D) Schematic of the STRAIGHT-IN procedure for simultaneously generating and identifying isogenic hiPSC clones for 12 different KCNH2 variants, along with the approximate time required for each step. (E) Dot plot of KCNH2 +/Acc hiPSCs transfected with the 12 KCNH2 variant donor vectors. Dots represent droplets containing the indicated sequence (attR or attP), while the percentage denotes the calculated integration efficiency. (F) Overview of the genomic sequence and location within KCNH2 of the introduced variant A561T, and sequence analysis from one of the resulting KCNH2 +/Acc-A561T hiPSC clones indicating the heterozygous introduction of c.G1681A. (G,H) Representative averaged field potential (FP) traces (G) and averaged FP duration (FPD) values (H) of KCNH2 +/Acc-wt and KCNH2 +/Acc-A561T hiPSC-CMs paced at 1.25 Hz. Colored arrowheads indicate the respective repolarization peak for each line. Values (n) indicate the number of recordings. Error bars represent ± SEM, and * p < 0.0001 (unpaired t-test).

Article Snippet: hiPSCs were transfected by lipofection with either an Flp-expressing plasmid (1.6 μg, pCAG_FlpoIRESpuro ( )), a Cre-expressing plasmid (1.6 μg, pEFBOS_CreIRESpuro ( )) or StemMACSTM Cre Recombinase mRNA (200 ng, Miltenyi Biotec).

Techniques: Binding Assay, Generated, Amplification, Expressing, Clone Assay, Transfection, Variant Assay, Sequencing

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