hdpp4 dna copy numbers  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus

doi: 10.1371/journal.pone.0145561

Figure Lengend Snippet: (A) Schematic diagram of the optimized hDPP4 expression vector cassette. The optimized hDPP4 was cloned into a pCAGGS plasmid in which hDPP4 expression was driven by the CAG promoter. hDPP4 expression was confirmed in vitro by transfection of Cos-7 cells with the pCAGGS-hDPP4 (B) or pCAGGS (C) plasmids and detected by direct immunofluorescence assay with FITC-labeled anti-human CD26–Fluorescein antibody. Confirmation of the binding between DPP4 and MERS-CoV RBD was achieved by transfecting Cos-7 cells with pCAGGS-hDPP4 (D) or pCAGGS (E) plasmid followed by indirect immunofluorescence assay with MERS-RBD-Fc protein and DyLight 549-conjugated goat anti-human IgG antibody. (F) Determination of the copy numbers of hDPP4 cDNA in four transgenic founder lines by qPCR. (G) Expression of hDPP4 mRNA in the indicated tissues of transgenic mice in two founder lines as determined by qRT-PCR. Results are mean±SEM ( n = 3). (H) Four lines of hDPP4 transgenic mice were infected with MERS-CoV and monitored for body weight changes. Results are mean±SEM ( n = 6). (I) Lung viral titer at day 5 postinfection was determined for four lines of hDPP4 transgenic mice. The data are expressed as mean±SEM ( n = 3). The dotted line indicates the limit of detection.

Article Snippet: The hDPP4 DNA copy numbers were determined by quantitative PCR using Power SYBR ® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA).

Techniques: Expressing, Plasmid Preparation, Clone Assay, In Vitro, Transfection, Immunofluorescence, Labeling, Binding Assay, Transgenic Assay, Quantitative RT-PCR, Infection

Journal: PLoS ONE

Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus

doi: 10.1371/journal.pone.0145561

Figure Lengend Snippet: The hDPP4 transgenic mice were infected intranasally with MERS-CoV. Lungs, kidneys, liver, spleen and brain were collected on day 5 or day 9 after MERS-CoV infection, fixed in neutral formaldehyde solution and stained with hematoxylin and eosin. Histopathological analysis of hDPP4 transgenic mice was performed using sham-infected mice as controls (A, D, G, J). (B-C) In the lungs, mild inflammation was observed, with inflammatory cell infiltration (arrowheads) and focal hemorrhage and exudation (arrow) on day 5. Damage was more severe on day 9, with increased inflammatory cell infiltration, focal hemorrhage (inset) and exudation. (E-F) In the kidney, MERS-CoV-infected hDPP4 transgenic mice showed focal interstitial inflammation with inflammatory cell infiltration in the interstitium and exudates in renal tubules (arrow) on day 5. On day 9, degeneration and necrosis in the renal tubular epithelial cells (inset) and focal hemorrhage were observed. (H-I) In the liver, MERS-CoV-infected hDPP4 transgenic mice showed scattered hepatocyte necrosis and numerous activated kupffer cells and infiltrated macrophages in the hepatic sinusoid on day 5. Fatty change of hepatocytes (inset) was observed in the liver on day 9. (K-L) In the spleen, necrotic splenic cells and increased reticulum cells in the red pulp with significant amounts of hemosiderin deposition were observed in hDPP4 transgenic mice on days 5 and 9 (arrow and inset). (M-O) Neurological damage with perivascular cuffs (M) and neuronal cell necrosis in the cerebral cortex (N) including damaged neurons (inset) in the hippocampus (O) was observed in the brains of hDPP4 transgenic mice infected with MERS-CoV. ( n = 2, scale bars = 50 μm).

Article Snippet: The hDPP4 DNA copy numbers were determined by quantitative PCR using Power SYBR ® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA).

Techniques: Transgenic Assay, Infection, Staining

Journal: PLoS ONE

Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus

doi: 10.1371/journal.pone.0145561

Figure Lengend Snippet: Transgenic mice were infected intranasally with MERS-CoV and sacrificed to access the expression of viral antigens by immunohistochemical staining ( n = 2). No viral antigens were detected in the sham-infected mice (A, D, G), but viral antigens were detected in type I and type II pneumocytes and infiltrated macrophages in the lungs (B-C), renal tubular epithelial cells in the kidneys (E-F), and neuron cell bodies as well as in dendrites and axons in the brain (H-I), including the hippocampus (I). (J) Viral load was detected by qRT-PCR and expressed as viral RNA copies/g of tissues. The detection limit shown as dotted line is 1×10 3 copies/g. Results are mean±SEM ( n = 2). (K) Viral titers in tissues of infected hDPP4 transgenic mice were determined by CPE-based assay and expressed as Log 10 TCID 50 /g of tissues. Results are mean±SEM ( n = 5). The dotted line indicates the detection limit.

Article Snippet: The hDPP4 DNA copy numbers were determined by quantitative PCR using Power SYBR ® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA).

Techniques: Transgenic Assay, Infection, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR