Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 1. Overview of the design and dual-functionality of the TRE-Lox system. (a) Overall structure of the 5′ end of the murine cathepsin D (CatD) gene (CTSD) and its promoter region (PCTSD, dark gray), indicating the relative position of the two gRNAs (black arrows) used for CRISPR/Cas9- assisted homologous recombination. Note the placement of the TATA box (TATA) very close to the main transcription start site (TSS) (right-angle arrow), the presence of the initiation codon (ATG, dashed white line) within Exon 1 (Ex 1, light gray), and the presence of a splice donor (SD) and splice acceptor (SA) flanking Intron 1 (black line). (b) Structure of the TRE-Lox knock-in (KI) insert, illustrating the relative positions of the two tet-operons (tetO2, green) and one LoxP site (LoxP, light blue) within the 5′ untranslated region (5′UTR) and, within Intron 1, a tetracycline response element (TRE) comprised of seven tetO repeats (tetO7) and the second LoxP site. The relative placement of the puromycin resistance cassette (Puror, purple) flanked by two FRT sites (FRT, dark blue), which is excisable by Flp recombinase, is depicted using a curly bracket. (c) Downregulation of CTSD via the action of rtTRKRAB acting on the TRE-Lox insert. In the presence of Dox (red triangles), rtTRKRAB binds to the tetO repeats within both the 5′UTR and Intron 1, triggering methylation of histones in a radius of 2–3 kb, thereby remodeling the chromatin and silencing the CTSD gene. (d) Genetic deletion of CTSD via the action of Cre recombinase on the TRE-Lox insert. The figure depicts the end result of Cre-mediated recombination of the TRE-Lox KI insert, which causes removal of the initiation codon, the first portion of the coding region of Exon 1 encoding the signal peptide of CatD, and the 5′ end of Intron 1.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: CRISPR, Homologous Recombination, Knock-In, Methylation

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 2. Development and characterization of TRE-Lox KI cell lines targeting the endogenous CTSD gene of mouse embryonic fibroblasts (MEFs). (a) Overview of the genotyping strategy, including the relative position of different primers (blue arrows) and the predicted sizes of different PCR amplicons (black boxes) used to distinguish different possible outcomes of insertion of the TRE-Lox construct via Cas9-assisted homologous recombination. The main possibilities include (but are not limited to) the following: (1) the unmodified endogenous murine CTSD allele (top); (2) insertion of the TRE-Lox KI insert as designed (middle); and (3) nonhomologous DNA end joining (NHEJ) resulting (in this case) in the excision of the segment of DNA between gRNA1 and gRNA2 (bottom, see Supplementary Materials Figure S1). (b) Genotyping of a subset of clones obtained after selection of individual puromycin-resistant clonal lines. The two bands within clone 1C8 (referred to as TL1C8) were excised, sequenced, and confirmed to be amplified from one allele carrying the TRE-Lox KI insert (upper band) and another allele featuring NHEJ, which results in functional knock-out (KO) of CTSD (sequences provided in Supplementary Materials Figure S5a–c). (c) CatD proteolytic activity in wild-type MEFs, or TL1C8 cells transiently transfected with empty vector (yellow), GFP (green) or Flp recombinase (dark blue). Note the low level of CatD activity in TL1C8 cells, which is reversed by transfection with Flp recombinase, resulting in activity close to the expected value of 50% of wild-type MEFs (gray dotted line). (d) CatD activity in several clonal lines of TL1C8-Flp cells stably expressing rtTRKRAB incubated for 4 d in the absence or presence of Dox (100 ng/mL). Note how, in the absence of Dox (white columns), all tested clones harbor CatD activity that is very close to 50% of the levels within MEFs (gray dotted line), as expected, whereas in the presence of Dox (gray columns), CatD activity is greatly decreased. Data in (c,d) are mean ± SEM of 4 replicates. * p < 0.05; ns = nonsignificant.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: Construct, Homologous Recombination, Clone Assay, Selection, Functional Assay, Knock-Out, Activity Assay, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Incubation

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 4. Functional and genotypic characterization of Cre-mediated genetic deletion of CatD made possible by the TRE-Lox system. (a) CatD activity in TL1C8-Flp cells transiently transfected with either GFP only (CTL, dark blue) or GFP-tagged Cre recombinase (Cre, green). Note that these are pools of GFP-positive cells selected by cell sorting 2 d after transfection. Data are mean ± SEM, n = 4,

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: Functional Assay, Activity Assay, Transfection, FACS

Journal: eLife

Article Title: A visual pathway for skylight polarization processing in Drosophila

doi: 10.7554/eLife.63225

Figure Lengend Snippet: ( A ) Confocal section of the medulla (dorsal view) showing R7/R8 photoreceptors (24B10 antibody staining: green) and their proximity to MeTu neurons (R56F07-Gal4>GFP: magenta). Right: Enlargement of medulla dorsal rim area (MEDRA), indicating contact between R7 and MeTu neurons. Scale bar denotes 10 μm. ( B ) Confocal projections of a single MCFO clone of R56F07 MeTu neurons with dendrites in the anterior/dorsal medulla (ME) in proximity to the medulla dorsal rim area. Left: Dorsal view. Center: Anterior view. Right: High-magnification projections showing the position of terminals in the anterior optic tubercle (AOTU), in anterior (top) and dorsal (bottom) view. Schematic indicates the position of the dendrites in the MEDRA and the terminals in the AOTU. ( C ) As in ( B ), for a MeTu neuron with dendrites in the mid/dorsal medulla. ( D ) As in ( B ), for a MeTu neuron with dendrites in the posterior medulla. Scale bars denote 10 μm.

Article Snippet: From confocal images of single-cell MCFO (MultiColor FlpOut) clones , we determined a consistent relationship between the anterior→posterior position of MeTu dendrites in the MEDRA and the ventral→dorsal position of MeTu axon terminals in the AOTU ( , ).

Techniques: Staining

Journal: eLife

Article Title: A visual pathway for skylight polarization processing in Drosophila

doi: 10.7554/eLife.63225

Figure Lengend Snippet: Experimental genotypes.

Article Snippet: From confocal images of single-cell MCFO (MultiColor FlpOut) clones , we determined a consistent relationship between the anterior→posterior position of MeTu dendrites in the MEDRA and the ventral→dorsal position of MeTu axon terminals in the AOTU ( , ).

Techniques: Comparison

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of GDF15 knockout mice on a high fat diet (HFD) . (A and B) Body weight and percent body weight gain of wild type (WT) and GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (C) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT), harvested at the end of the study, 25 weeks of HFD (n = 13,9). (D) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 14,9). (E) Lipid droplet area (Percent liver area) determined from histological analyses of haematoxylin/eosin (H&E) stained liver sections (n = 9,8). (F) Plasma triglycerides (TG), cholesterol, leptin, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice after 16 weeks of HFD-feeding (n = 12,11). (G) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice, after 16 weeks of HFD feeding (n = 16–29). (H and I) Blood glucose levels during intraperitoneal (ip) glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (J) Plasma FGF21 levels from random fed mice at 16 weeks of HFD feeding (n = 7,9). (K) FGF21 mRNA expression in tissues from WT and GDF15 KO mice after 25 weeks HFD feeding (n = 8–11). All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Staining, Clinical Proteomics, Expressing

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: GDF15 mRNA expression within high fat diet fed mouse and human adipose tissue . (A) In situ hybridization analysis of GDF15 mRNA (red) from 18-week-old high fat diet (HFD) fed wild type (WT) mouse epididymal adipose tissue. Black arrows indicate GDF15 staining in foamy macrophages contributing to the formation of ‘crown-like structures’. Blue arrow indicates GDF15 staining within adipocytes. (B) Representative haematoxylin/eosin image of epididymal tissue from 18-week old HFD fed WT mouse. Black arrows indicate infiltrating cells. (C) Representative image (corresponding to the image in B) of epididymal tissue from 18-week-old HFD fed WT mouse stained with the macrophage marker F4/80 confirming that the cells contributing to the ‘crown like structures’ are macrophages. (D and E) GDF15 and EMR1 mRNA expression from 14 week old Chow or HFD fed WT mouse epididymal tissue fractionated into adipocytes, CD11b negative (−) and CD11b positive (+) stromal vascular fractions (SVF) (n = 3–4). (F) Correlation of GDF15 expression with EMR1 expression in epididymal tissue from 14 week Chow or HFD fed wild type mice (n = 8). (G–I) Human subcutaneous adipose tissue GDF15 gene expression levels in the TwinsUK adipose study associated with estimated macrophage proportion in adipose tissue (G), and macrophage markers CD68 and EMR1 (H and I). Each point represents data from a single individual. Plotted gene expression residuals of GDF15, CD68 and EMR1 were adjusted for age, BMI and RNA-Seq technical covariates. (J and K) GDF15 gene expression levels in the ‘Obese study’ associated with macrophage marker CD68 in human subcutaneous and visceral adipose tissue. (L and M) In situ hybridization analysis of Gdf15 mRNA (red) from 18-week old HFD fed WT mouse liver and brown adipose tissue. (N) FGF21 mRNA expression from 14 week Chow or HFD fed WT mouse epididymal tissue fractionated in to adipocytes, CD11b negative (−) and CD11b positive (+) stromal vascular fraction (SVF) (n = 3–4).

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Expressing, In Situ Hybridization, Staining, Marker, Gene Expression, RNA Sequencing

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of LysM-Cre mediated GDF15 macrophage knockout mouse on a high fat diet (HFD) . (A) GDF15 mRNA expression from 24-week-old high fat diet (HFD) fed wild type (WT) and LysM-GDF15 KO mouse epididymal adipose tissue fractionated into adipocytes and CD11b positive (+) stromal vascular fraction (SVF) (n = 4). (B) GDF15 mRNA expression in tissues from 24 week HFD fed WT and LysM-GDF15 KO mice. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), liver and kidney (n = 4–7). (C) Plasma GDF15 levels from random fed mice at indicated weeks on HFD (n = 15–18). (D) FGF21 mRNA expression in tissues from 24-week HFD fed WT and LysM-GDF15 KO mice (n = 4–7). (E) Plasma FGF21 levels from random fed mice at indicated weeks on HFD (n = 11–18). (F and G) Body weight and percent body weight gain of WT and LysM-GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (H) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 16–18). (I) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 16,17). (J) Plasma leptin levels in mice from random fed mice after 16 weeks HFD-feeding (n = 16–18). (K) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice, after 16 weeks of HFD feeding (n = 16,18). (L and M) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (N) Plasma triglycerides (TG), cholesterol, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice, after 16 weeks of HFD feeding (n = 9–11). All data are means ± S.D ∗/∗∗ - p < 0.05/0.01.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of bone marrow deleted GDF15 knockout mouse on a high fat diet . (A) GDF15 mRNA expression in tissues from 24-week old high fat diet (HFD) fed WT and BMT-GDF15 KO mice. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT) and brown adipose tissue (BAT) (n = 8–12). (B) Plasma GDF15 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 12). (C) FGF21 mRNA expression from indicated tissues of 24 week HFD fed WT and BMT-GDF15 KO mice (n = 8–12). (D) Plasma FGF21 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 12). (E and F) Body weight and percent body weight gain of WT and BMT-GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (G) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 3–12). (H) Plasma triglycerides (TG), cholesterol, leptin, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice, after 16 weeks of HFD feeding (n = 12). (I) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 12). (J) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice after 16 weeks of HFD feeding (n = 7,6). (K and L) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of Alb-Cre mediated GDF15 hepatocyte knockout mouse on a high fat diet . (A) GDF15 mRNA expression in tissues from 24-week old high fat diet (HFD) fed wild type (WT) and Alb-GDF15 KO mice. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver, brown adipose tissue (BAT) and kidney (n = 10–16). (B) Plasma GDF15 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 15,13). (C) FGF21 mRNA expression in indicated tissues from 24-week HFD fed WT and Alb-GDF15 KO mice (n = 10–13). (D) Plasma FGF21 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 15,13). (E and F) Body weight and percent body weight gain of WT and Alb-GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (G) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 15,11). (H) Plasma leptin, triglycerides (TG), cholesterol, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice, after 16 weeks of HFD feeding (n = 15,13). (I) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 15,16). (J and K) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (L) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice, after 16 weeks of HFD feeding (n = 14,13). All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of dKO knockout mice on a high fat diet . (A) Liver GDF15 and (B) FGF21 mRNA expression from 24-week-old high fat diet (HFD) fed WT, FGF21 KO, GDF15 KO and FGF21/GDF15 double knockout (dKO) mice (n = 5). (C and D) Plasma GDF15 and FGF21 from random fed mice at indicated time after high fat feeding (n = 3–11). (E and F) Body weight and percent body weight gain of WT, FGF21 KO, GDF15 KO and dKO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (G and H) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (I) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice after 16 weeks of HFD feeding (n = 19–29). (J) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 11–17). (K) Weight of total hepatic lipids; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 9–17). (L) Lipid droplet area (Percent liver area) determined from histological analyses of H&E stained liver sections (n = 8–11). (M) Plasma triglycerides (TG) from random fed mice, after 24 weeks of HFD feeding (n = 8). (N) Hepatic mRNA expression of genes involved in lipid metabolism (n = 9–17). All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001. Please note that the data for WT and GDF15KO overlaps with the data presented in Figure 1 .

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Double Knockout, Clinical Proteomics, Staining

Journal: eLife

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.7554/eLife.70948

Figure Lengend Snippet: ( A ) Schematic of the targeting strategy to generate the T6B mouse. The construct contains a flippase recognition target site (frt) that allows homing into the Col1a1 locus when electroporated together with a vector expressing the Flippase recombinase into KH2 ( Col1a1-frt/Rosa26-rtTA ) murine embryonic stem cells. KH2 also express the rtTA trans-activator driven by the endogenous Rosa26 (R26) promoter. ( B ) Immunofluorescence imaging performed using an anti-YFP antibody, showing T6B expression in a panel of tissues of adult R26 T6B mice fed doxycycline for 7 days. Tissues from R26 CTL (carrying the rtTA allele but not the T6B allele) were used as negative controls. ( C ) Protein lysates from the liver of R26 T6B mice on or off doxycycline-containing chow for the indicated number of days were resolved by SDS-PAGE and western blotting was performed with anti-HA antibody to detect expression of the T6B transgene. ( D ) Co-IP experiments using an anti-YFP antibody showing interaction between AGO and T6B in total liver extracts from T6B mice on doxycycline-containing chow. ( E ) Size-exclusion chromatography (SEC) elution profile of AGO2-containing complexes in liver lysates from T6B mice euthanized at the indicated time points after doxycycline administration. Notice the shift of AGO2 from the high-molecular-weight fractions to the low-molecular-weight fractions after 5 days of doxycycline treatment and the reconstitution of the full miRISC after removal of doxycycline from the diet. ( F, G ) Total RNA extracted from the large intestine ( F ) and the liver ( G ) of R26 CTL and R26 T6B mice was subjected to RNAseq (n = 3 for each strain). Left panel: scatter plot showing the effect of T6B expression on targets of all miRNA families was generated as described in . The abundance of each miRNA family was calculated using dataset from . Right panel: representative cumulative distribution plot of log2-fold changes in expression of predicted targets of the indicated miRNA families. Figure 2—source data 1. RNAseq, differential gene expression, colon and liver. Figure 2—source data 2. Z-scores and miRNA families abundance, colon and liver. Figure 2—source data 3. Unedited blots shown in . Figure 2—source data 4. Uncropped blots shown in . Figure 2—source data 5. Unedited blots shown in . Figure 2—source data 6. Uncropped blots shown in . Figure 2—source data 7. Unedited blots shown in . Figure 2—source data 8. Uncropped blots shown in .

Article Snippet: Recombinant DNA reagent , pCAGGS-flpE-puro (plasmid) , Addgene , RRID: Addgene_20733 , Flippase recombinase- expressing vector.

Techniques: Construct, Plasmid Preparation, Expressing, Immunofluorescence, Imaging, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Size-exclusion Chromatography, High Molecular Weight, Molecular Weight, Generated, Gene Expression

Journal: eLife

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.7554/eLife.70948

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCAGGS-flpE-puro (plasmid) , Addgene , RRID: Addgene_20733 , Flippase recombinase- expressing vector.

Techniques: Marker, Expressing, Irradiation, Transfection, Construct, Negative Control, Control, Recombinant, Plasmid Preparation, Sequencing, Mutagenesis, RNAscope, Software, Knock-Out, Staining, Amplification

Journal: Neuron

Article Title: Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

doi: 10.1016/j.neuron.2019.01.035

Figure Lengend Snippet: Figure 5. Characterization and Use of an LSL-Cas9 Transgenic Rat for Cre-Dependent Knockout of TH (A) Representative confocal images of colocalization of iCre recombinase and the FLAG-tagged Cas9 transgene in LSL-Cas9 rats. FLAG immunoreactivity is not observed following delivery of Flpo, a non-Cre recombinase. iRFP fluorescence indicates comparable delivery of Flpo- and iCre-encoding viruses. (B) Representative confocal images of unilateral TH loss in the SN of LSL-Cas9 rats 4 weeks after a midbrain injection of AAV iCre and AAV control gRNAs (right side, top) or AAV Th gRNAs (left side, bottom). Comparable EGFP fluorescence in control and Th gRNAs-injected hemispheres indicates comparable viral delivery between conditions. (C) A TH-immunostained striatal section from a rat injected as in (B) (L, left side; R, right side). (D) Quantification of optical density of TH immunoreactivity in the SN and striatum of animals described in (B) and (C). Each data point represents one analyzed coronal section (n = 3–4 sections/animal), and each color represents a different animal (n = 4). Scale bars represent 50 mm (A), 100 mm (B), and 500 mm (C).

Article Snippet: The AAV vectors expressing the iRFP713 reporter along with iCre or Flpo recombinases (Addgene 112683 and 112684) were constructed by using the coding region for iRFP713 tagged with a nuclear localization signal to replace the eGFP cassette in pAAV CMVIE eGFP-2A-iCre and pAAV CMV-IE eGFP-2A-FLPo.

Techniques: Transgenic Assay, Knock-Out, Injection, Control

Journal: Neuron

Article Title: Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

doi: 10.1016/j.neuron.2019.01.035

Figure Lengend Snippet: Figure 6. Developing an Assay for Knockout of MANF In Vivo (A) A schematic of the gRNA-binding sites and the PCR assay used to amplify the 893 nt flanking the second exon of rat Manf. (B–E) Rat primary cortical neurons were transduced with AAV Cas9 and AAV Manf gRNAs or AAV control gRNAs and harvested 1 week later for determination of mutagenesis and knockout. (B) Co-transduction with AAV Cas9 and AAV Manf gRNAs resulted in resolvase-induced cleavage of the PCR product (arrows). (C) An alignment of seven independently isolated clones of the PCR fragment shows precise +A insertions among the alleles. Knockout of Manf was verified with (D) real- time qRT-PCR and (E) Wes analyses of Manf mRNA and protein levels, respectively. (D) Manf mRNA levels were normalized to the geometric mean of reference genes and presented as 2ddCq ± upper and lower limits (n = 3, unpaired t test using dCq values, t(4) = 20.27, ****p < 0.0001). (E) The MANF protein band density was normalized to actin and presented as density relative to control gRNA (mean ± SE, n = 3, unpaired t test, t(4) = 4.999, **p = 0.0075). The arrow in the cropped blot points at the 25-kDa MANF band. (F) Representative images of unilateral loss of MANF immunoreactivity in the SN of LSL-Cas9 rats four weeks after co-injection of AAV iCre and AAV control gRNAs or AAV Manf gRNAs. GFP fluorescence represents delivery of gRNA. Scale bar 100 mm. (G) Quantification of the optical density of MANF immunoreactivity in the SN of LSL-Cas9 or WT animals injected as described in (F). Each data point represents one analyzed coronal section (n = 3–4/animal), and each color represents data from a distinct animal (n = 4/group, unpaired t test, t(6) = 5.437, **p = 0.0016).

Article Snippet: The AAV vectors expressing the iRFP713 reporter along with iCre or Flpo recombinases (Addgene 112683 and 112684) were constructed by using the coding region for iRFP713 tagged with a nuclear localization signal to replace the eGFP cassette in pAAV CMVIE eGFP-2A-iCre and pAAV CMV-IE eGFP-2A-FLPo.

Techniques: Knock-Out, In Vivo, Binding Assay, Transduction, Control, Mutagenesis, Isolation, Clone Assay, Quantitative RT-PCR, Injection