#3013017- CASTLE NUT- JT15 PRATT WHITNEY ENGINE
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Schematic diagram of pronuclear injection-based targeted transgenesis (PITT).
The donor vector is co-injected with Cre expression plasmid into the pronuclei of fertilized eggs obtained from seed mice. As a result of site-specific recombination between mutant loxPs (lox2272, JTZ17 and JT15) by Cre enzyme, one copy of the donor vector is integrated into Rosa26 genomic locus to yield recombination intermediate mice. Extra sequence containing selection marker (Neo) and vector sequence is removed by flippase (FLP)–FLP recombinase target (FRT) gene rearrangement, resulting in generation of Nephrin-EGFP mice. Nephrin, nephrin promoter sequence; EGFP, EGFP cDNA; pA, poly A signal; Neo, neomycin resistance gene cassette; FLPe, FLPe-overexpressing deleter mouse.
Primer and probe sequences for qPCR of genomic DNA and cDNA.
EGFP expression and genotyping of Nephrin-EGFP mice.
(A) EGFP protein was expressed preferentially at periphery of glomeruli in two lines of Nephrin-EGFP mice by immunohistochemistry using anti-GFP antibody. (B) Genotyping of wild-type (WT), heterozygous (Hetero) and homozygous (Homo) mice by genomic qPCR. Amount of EGFP genomic DNA detected in WT mice was less than 1% of heterozygous mice. Mean±SEM of n = 4–8. Comparison was carried out by unpaired t test. Magnification, 10x.
Protein expression of EGFP and glomerular cell markers in Nephrin-EGFP mice.
(A-C) EGFP and nephrin were co-expressed along glomerular capillaries (yellow arrowheads). Cell bodies of podocytes located at the outer surface of glomerular capillaries (green arrowheads) also expressed EGFP (A) but nephrin expression was spared (B). (D) Similarly, EGFP and podocin were expressed along glomerular capillaries (yellow arrowheads). Podocyte cell bodies expressed EGFP but not podocin (green arrowheads). (E) PDGFRβ was expressed at glomerular and peritubular capillaries (arrowheads) and walls of arterioles (arrows), and did not co-localize with EGFP. (F) PECAM-1 was expressed along glomerular and peritubular capillaries (arrowheads) and inner surface of arterioles (arrow), and did not merge with EGFP. Magnification, 40x.
EGFP expression in cultured glomeruli from Nephrin-EGFP mice.
On Day 2, EGFP expression remained mainly along the periphery of glomeruli, but was markedly reduced on day 5. FBS concentration was reduced from 10% to 0.5% on day 3. Treatment with 1α,25-(OH)2 vitamin D3 (VD, 50 nM) and all trans-retinoic acid (RA, 1 μM) for the last 2 days induced EGFP expression. Veh, vehicle. Magnification, 10x.
Expression of nephrin and podocin mRNA in cultured glomeruli.
Gene expression was studied on days 0–5 (D0-D5) and at 6 hours (6h). Vitamin D (VD, 50 nM) or vehicle (Veh) was added for the last two days. Expression levels were normalized by 18S ribosomal RNA and levels at D0 were defined as 100%. Mean±SEM of n = 5. Treatment with VD significantly upregulated nephrin expression by unpaired t test. NS, not significant. *P
Quantitative evaluation of EGFP signals in glomerular areas.
(A) Glomeruli appeared as clusters of DAPI signals. (B) DAPI clusters having areas larger than > 2000 μm 2 were recognized as glomeruli and ROI were set to such glomerular areas (yellow arrows), whereas smaller clusters were excluded from ROI (green arrows). (C) EGFP signals were measured only in ROI. Magnification, 10x.
Histogram of EGFP signals of respective glomeruli.
(A) In a single well, total EGFP signal of each glomerulus was calculated individually, and fractionated as histogram. (B) EGFP signal per DAPI signal was calculated and analyzed. (C) Each bar indicates mean±SEM of EGFP/Area among several dozens of glomeruli from a single well (Fig 8A). Vitamin D+retinoic acid (VD+RA) or vehicle (Veh)-treated glomeruli were studied in triplicate wells. (D) Each bar indicates mean±SEM of EGFP/DAPI of a well.
Effects of FBS upon EGFP signals.
Glomeruli from Nephrin-EGFP mice were cultivated for the first 3 days (D0-D3) and the last 2 days (D4-D5) with DMEM containing different concentrations of FBS and fluorescence was examined on D5. Vitamin D (50 nM, VD)+retinoic acid (1 μM, RA) or vehicle (Veh) was added during D4-D5. (A) Glomerular count per well of glomeruli given VD+RA during D4-D5. Mean±SEM of n = 3. (B) EGFP signal per glomerular area. N = 3. (C) Effects of VD+RA upon EGFP/Area signals. The average value from wells treated with VD+RA was divided by the average value from Veh-treated wells.
Dose-dependent induction of EGFP signals and nephrin mRNA expression by vitamin D.
Cultured glomeruli from Nephrin-EGFP mice were treated with 1–1250 nM of vitamin D (VD), 1 μM retinoic acid (RA) or vehicle (0 nM) for the last 2 days and analyzed on day 5. (A) EGFP/glomerular area. Mean background signal of wild-type glomeruli was subtracted. (B) Nephrin mRNA expression. Expression levels were normalized by 18S ribosomal RNA. Each bar indicate mean±SEM of triplicate wells. The level of vehicle was defined as 1.0 unit. # P
Effects of DMSO upon EGFP signals.
Vitamin D (VD) or DMSO was added for the last 2 days and fluorescence was examined as EGFP/glomerular area on day 5. Mean±SEM of n = 3. Addition of 2% DMSO significantly decreased EGFP signals both in vehicle- and VD-treated glomeruli (P<0.05). Treatment with VD significantly increased EGFP signals in the presence of all concentrations of DMSO studied (0 to 2%, P<0.001). Comparison was carried out by two-way ANOVA with Bonferroni’s multiple comparison test.
Quantitative evaluation of changes in EGFP, nephrin and podocin protein expression in glomeruli by vitamin D.
Endogenous nephrin and podocin were visualized by immunofluorescence using primary antibodies and Alexa Fluor 568-conjugated secondary antibodies. Fluorescence intensity was measured on days 1–5 (D1-D5). Glomeruli were treated with 50 nM vitamin D (VD) or vehicle (Veh) for the last 2 days and examined on day 5 (D5), and the difference was studied by unpaired t test. For EGFP quantitation, mean background signal of wild-type glomeruli was subtracted and the level of Veh-treated Nephrin-EGFP glomeruli on D5 was defined as 1.0 unit. For nephrin and podocin, mean background signal of wild-type glomeruli incubated with secondary antibody alone was subtracted, respectively. Difference between EGFP signals among days was examined by one-way ANOVA with Bonferroni’s multiple comparison test. N = 5.
An example of chemical library screening of a plate.
Y axis indicates EGFP intensity/glomerular area of each well under excitation. Wells treated with vitamin D (red circles) or vehicle (blue circles) are highlighted.
Strong and linear effects by auto-fluorescence of chemicals upon screening by EGFP signal intensity.
(A) Cat#, catalog number. a, Pairs of compound concentrations with linear dose-response (10-times more compound giving approximately 10-times more signal), which suggest non-specific effects reflecting compound colors. b, Glomeruli treated with 10 μM of Ro-31-8220 were detached from plate likely due to drug toxicity. c, 10 nM Vitamin D and 1% DMSO served as positive and negative controls for EGFP/glomerular area signals under excitation. Background subtraction of signal by wild-type glomeruli was not carried out here. (B) Photograph of chemical compound stock solution (1 mM) under standard room light, showing intrinsic colors in some solutions.