Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice

BACKGROUND: Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. RESULTS: The GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney. CONCLUSION: The goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals

Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

<p>Abstract</p> <p>Background</p> <p>Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD₅₀of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.</p> <p>Results</p> <p>Secretion level of the fusion protein produced <it>in vitro </it>in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced <it>in vitro </it>was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). <it>In vitro </it>nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.</p> <p>Conclusion</p> <p>Both the pharmacokinetic study and the <it>in vitro </it>nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced <it>in vitro </it>and <it>in vivo</it>. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.</p

Relationship of Weather Types on the Seasonal and Spatial Variability of Rainfall, Runoff, and Sediment Yield in the Western Mediterranean Basin

Rainfall is the key factor to understand soil erosion processes, mechanisms, and rates. Most research was conducted to determine rainfall characteristics and their relationship with soil erosion (erosivity) but there is little information about how atmospheric patterns control soil losses, and this is important to enable sustainable environmental planning and risk prevention. We investigated the temporal and spatial variability of the relationships of rainfall, runoff, and sediment yield with atmospheric patterns (weather types, WTs) in the western Mediterranean basin. For this purpose, we analyzed a large database of rainfall events collected between 1985 and 2015 in 46 experimental plots and catchments with the aim to: (i) evaluate seasonal differences in the contribution of rainfall, runoff, and sediment yield produced by the WTs; and (ii) to analyze the seasonal efficiency of the different WTs (relation frequency and magnitude) related to rainfall, runoff, and sediment yield. The results indicate two different temporal patterns: the first weather type exhibits (during the cold period: autumn and winter) westerly flows that produce the highest rainfall, runoff, and sediment yield values throughout the territory; the second weather type exhibits easterly flows that predominate during the warm period (spring and summer) and it is located on the Mediterranean coast of the Iberian Peninsula. However, the cyclonic situations present high frequency throughout the whole year with a large influence extended around the western Mediterranean basin. Contrary, the anticyclonic situations, despite of its high frequency, do not contribute significantly to the total rainfall, runoff, and sediment (showing the lowest efficiency) because of atmospheric stability that currently characterize this atmospheric pattern. Our approach helps to better understand the relationship of WTs on the seasonal and spatial variability of rainfall, runoff and sediment yield with a regional scale based on the large dataset and number of soil erosion experimental stations

Tissue extracts from a transgenic mouse (99-120-2F) and a negative control mouse (99-120-6F) were separated on 4–20 % SDS-PAGE and transferred onto the membrane

<p><b>Copyright information:</b></p><p>Taken from "Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice"</p><p>BMC Biotechnology 2005;5():9-9.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1090560.</p><p>Copyright © 2005 Huang et al; licensee BioMed Central Ltd.</p> The GFP positive control was from a cell line with GFP expression. 12 μg of total protein was loaded in each lane. Immunoreacting bands were detected using a polyclonal anti-GFP antibody (Clontech). . The same blot was reprobed with a mouse monoclonal anti-actin antibody (Chemicon) to show that equal amount of protein was loaded in each lane

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice-5

<p><b>Copyright information:</b></p><p>Taken from "Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice"</p><p>BMC Biotechnology 2005;5():9-9.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1090560.</p><p>Copyright © 2005 Huang et al; licensee BioMed Central Ltd.</p>structures with thick walls indicative of the thick segment of the ascending limb of Henle's loop within the TAL region. Fixed kidney sections from a negative control mouse () and the 99-122-1-A5M transgenic mouse () were stained with a polyclonal anti-human uromodulin antibody, followed by treatment with Texas-Red conjugated secondary anti rabbit IgG antibody. Expression of endogenous uromodulin in the cells (red) appears in a punctuated pattern, whereas the GFP expression is cytoplasmic (green). Immunostaining of similar sections for uromodulin confirmed that expression is restricted to tubular epithelial cells of these structures () indicated by red. The same sections observed for GFP expression by confocal microscopy indicated that expression was restricted to similar structures as in &but not co-expressed with the endogenous uromodulin staining. As expected GFP was absent in the renal sections obtained from a non-transgenic mouse. Both GFP () (green) and uromodulin () (red) were co-expressed in cells in the TAL segment (transgenic mouse, 99-122-1-A5M)

Kidney extracts (12 μg of total protein loaded in each lane) from 99-120-2 and 99-122-1A3 were used as positive controls, whereas 99-120-6 was used as a negative control

<p><b>Copyright information:</b></p><p>Taken from "Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice"</p><p>BMC Biotechnology 2005;5():9-9.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1090560.</p><p>Copyright © 2005 Huang et al; licensee BioMed Central Ltd.</p> GFP immunoreacting protein was detected using the polyclonal anti-GFP antibody (Clontech) in 2 out 4 Day 0 pups, offspring of a transgenic F2 mouse, 00-074-1A2B8M, bred with a wild type mouse