Journal: bioRxiv

Article Title: SFPQ-TFE3 gene fusion reciprocally regulates mTORC1 activity and induces lineage plasticity in a novel mouse model of renal tumorigenesis

doi: 10.1101/2024.11.21.624702

Figure Lengend Snippet: (A) Dual IHC for PAX8 (brown) and GPNMB (teal) in tamoxifen-injected, SFPQ-TFE3 LSL ; Pax8-CreERT transgenic mice and age-matched, littermate controls, at 2 weeks following injection of tamoxifen. Scale bar = 100 µm. (B) H&E (top row) and dual PAX8/GPNMB IHC staining (bottom row) of kidneys from representative tamoxifen-injected, SFPQ-TFE3 LSL ; Pax8-CreERT transgenic mice and age-matched, littermate controls, at 3.5 months following injection of tamoxifen. Scale bar= 100 µm. (C) H&E (top row), dual PAX8/GPNMB IHC staining (middle row) and PAX2 IHC staining (bottom row) of kidneys from representative age-matched, control and SFPQ-TFE3 LSL ; Ksp-Cre transgenic mice at post-natal day 15. Scale bar = 100 µm. (D) H&E (top row), dual PAX8/GPNMB IHC staining (middle row) and PAX2 IHC staining (bottom row) of kidneys from representative age-matched, control and PRCC-TFE3 LSL ; Ksp-Cre transgenic mice at 7 to 9 months. (E) Digital quantification of mean nuclear PAX8 H-scores in GPNMB-cells (blue) and GPNMB+ cells (red) at day 1 and day 15 (Fix Figure), from experiments in C , as depicted by scatter plots, with the central line denoting the median. The number of biological replicates analyzed for both, GPNMB – and + cells were n=11 (day 1) and n=9 (day 15). Statistical analyses were performed using two-tailed Mann-Whitney test. (F) Digital quantification of mean nuclear PAX8 H-scores in GPNMB-cells (blue) and GPNMB+ cells (red) at 4 months and 7 months, from experiments in D , as depicted by scatter plots, with the central line denoting the median. The number of biological replicates analyzed for both, GPNMB – and + cells were n=9 at 4 and 7 months. Statistical analyses were performed using two-tailed Mann-Whitney test. (G) Gene Set Enrichment Analysis (GSEA) comparing 15-day STK (top panels), 3.5-month tamoxifen-treated, STP (middle panels) and 7-month PTK (bottom panels), transgenic kidneys and their controls, for genes differentially expressed in renal TSC-related PEComas from . (H) Immunoblotting of lysates from primary renal tubular epithelial cells from SFPQ-TFE3 LSL transgenic mice treated with control or Cre-recombinase expressing adenovirus in vitro for the indicated markers. Source data are provided as a Source data file.

Article Snippet: 2) Mice hemizygous for the Ksp -Cre recombinase knockin gene (Strain Number: 012237) (The Jackson Laboratory).

Techniques: Injection, Transgenic Assay, Immunohistochemistry, Control, Two Tailed Test, MANN-WHITNEY, Western Blot, Expressing, In Vitro

Journal: Vitamins and hormones

Article Title: GROWTH HORMONE RECEPTOR GENE DISRUPTION

doi: 10.1016/bs.vh.2022.12.004

Figure Lengend Snippet: Summary of thirty-seven GHR gene disrupted mouse lines.

Article Snippet: Both of these mouse lines were produced by the Kopchick laboratory at Ohio University by crossing the Kopchick floxed GHR mice with the ubiquitously-expressed ROSA26 gene promotor/enhancer-driven Cre-recombinase mice (B6.129- Gt(ROSA)26Sor tm1(Cre/ERT2)Tyj /J,Stock#008463, The Jackson Laboratories).

Techniques: Injection, Virus, Plasmid Preparation

Journal: Nature

Article Title: Calcium-permeable AMPA receptors govern PV neuron feature selectivity

doi: 10.1038/s41586-024-08027-2

Figure Lengend Snippet: a , To enable robust expression of eGFP-GluA2, we used a strong ubiquitous CMV-βactin hybrid (CAG) promoter (consisting of three gene regulatory elements: 5′ cytomegalovirus early enhancer element, chicken β-actin promoter and rabbit β-globin intron) and added a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) at the 3′ end of the eGFP-GluA2 coding sequence. The WPRE sequence allows rapid exit of mRNA from the nucleus and increases the mRNA stability in the cytosol. For inducible expression of eGFP-GluA2, a “stopper” cassette consisting of loxP-flanked 3X SV40 polyA (loxP-STOP-loxP, “lsl”) was placed upstream of the coding sequence, preventing expression until cyclic recombinase (Cre)-dependent excision. A Neomycin resistance cassette (Neo R ) flanked by Flippase Recognition Target sequences (FRT) was present in the targeting vector to allow for selection. To prevent gene-silencing effects and ensure consistent and long-term expression of these transgenes in all cell types, the CAG-driven inducible eGFP-GluA2 transgenic constructs were targeted to the ubiquitously expressed Rosa26 locus. For homologous recombination in mouse embryonic stem (ES) cells, the gene-targeting vector was assembled into a ROSA26 targeting plasmid containing a 1.2 kb 5′ homology arm, 4.3 kb 3′ homology arm, and PGK-DTA (Diphtheria toxin fragment A, downstream of 3′ homology arm) for negative selection. ES cells, derived from a SV129 mouse strain, were electroporated with the AsiSI-linearized targeting vectors. A nested PCR screening strategy along the 5′ homology arm was used to identify ES cell clones harboring the correct genomic targeting event. After verification of homologous recombination by Southern blot analysis and confirmation of the karyotypes, correctly targeted ES cell clones were used to generate chimeric mice by injection into blastocysts derived from SV129 females at the Johns Hopkins University Transgenic Core. Germline transmission was achieved by breeding male chimeric founders to C57BL/6 N wild-type female mice. The FRT-NeoR cassette was removed by breeding to a transgenic FLPe mouse line . b-j , Transgenic expression of GluA2 in PV interneurons mimics excitatory neuron GluA2 expression levels (related to Fig. ). b-d , Representative data for Fig. . Immunohistochemical staining of GluA2 expression in PV interneurons and excitatory neurons. PV interneurons are marked by white asterisks and display negative CaMKIIα staining. Images were acquired in layer 2/3 of visual cortex. Scale bars, 15 μm. e - g , Representative data for Fig. . Immunohistochemical staining of GluA1 expression in PV interneurons and excitatory neurons. Scale bars, 15 μm. h-j, Concordance of conditionally expressed eGFP and eGFP-GluA2 with PV immunostaining. h, i , Immunohistochemical staining of PV interneurons in mouse visual cortex. Scale bars, 100 μm. j , Quantification of conditional expression concordance in PV interneurons. In both PV-Cre;lsl-eGFP and PV-Cre; lsl-eGFP-GluA2 mouse lines, the ratio of PV+ cells among GFP+ cells and GFP+ cells among PV+ cells was high (n = 6/6 slices for each genotype; PV-Cre;lsl-eGFP mice: PV + /GFP + = 93.8 ± 2.7%, GFP + /PV + = 78.3 ± 8.3%; PV-Cre;lsl-eGFP-GluA2 mice: PV + /GFP + = 85.0 ± 5.1%, GFP + /PV + = 90.7 ± 4.0%). Bars and error bars denote mean ± SEM.

Article Snippet: We generated the FUW-Cre construct by replacing the eGFP in FUGW with the Cre recombinase gene. pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40 (ref. ) was a gift from D. Kim and the GENIE Project (Addgene, plasmid 100853).

Techniques: Expressing, Virus, Sequencing, Plasmid Preparation, Selection, Transgenic Assay, Construct, Homologous Recombination, Derivative Assay, Nested PCR, Clone Assay, Southern Blot, Injection, Transmission Assay, Immunohistochemical staining, Staining, Immunostaining