adenoviral vectors ad cre gfp  (Vector Biolabs)

Journal: The Journal of physiology

Article Title: Diet-induced obesity in mice increases carotid body chemosensitivity via the leptin-TRPM7 pathway

doi: 10.1113/JP288722

Figure Lengend Snippet: Confocal images showing Ad-Cre-GFP expression, suggesting successful transfection of the Ad-Cre-GFP viruses into the CB of diet-induced obese (DIO) Trpm7 flox mice ( N = 2). B , summary data showing relative expression ratio (RER) of Trmp7 mRNA in the carotid bodies of lean Trmp7 flox mice treated with control Ad-GFP ( N = 6) and Ad-Cre-GFP viruses ( N = 4). There was a significant decrease in the Trpm7 mRNA expression in the Ad-Cre-GFP group ( P = 0.002; Mann–Witney U test).

Article Snippet: Adenoviral vectors Ad-Cre-GFP containing Cre-recombinase tagged with green fluorescent protein (GFP, 3.3 × 10 12 VP/ml) and control Ad-GFP (3.3 × 10 12 VP/ml) were from Vector Biolabs (Malvern, PA, USA).

Techniques: Expressing, Transfection, Control

Journal: The Journal of physiology

Article Title: Diet-induced obesity in mice increases carotid body chemosensitivity via the leptin-TRPM7 pathway

doi: 10.1113/JP288722

Figure Lengend Snippet: A and B are representative illustrations of ex vivo CSN responses from diet-induced obese Trpm7 flox mice transfected into the CB with Ad-Cre-GFP and control Ad-GFP viruses, respectively. CSN responses were normalized to activity in hyperoxia (Hypx, PO 2 = 200 Torr, represented by the horizontal dashed line). Genetic knockdown of Trpm7 receptors completely abolished the stimulatory effect of leptin on CSN activity ( A ), whereas the leptin’s effect was intact when the Trpm7 channels were functional. B , the P -values for the effect of Trpm7 knockdown are shown in the figures C and D . C , summary data of basal CSN activity: normoxia (Nx) + Ad-Cre-GFP vs . Nx + leptin + Ad-Cre-GFP ; P = 0.148 ( N = 5), Nx + Ad-GFP vs . Nx + leptin + Ad-GFP ; P = 0.001 ( N = 5). D , the hypoxic chemoreflex represented as ΔCSN activity (Hx–Nx): Ad-Cre-GFP vs. Ad-Cre-GFP + leptin; P = 1.000, Ad-GFP vs. Ad-GFP + leptin; P < 0.001, Ad-Cre-GFP + leptin vs. Ad-Cre-GFP + leptin + FTY720; P = 0.456; Ad-GFP + leptin vs. Ad-GFP + leptin + FTY720; P = 0.001 ( N = 5). There was a statistically significant interaction between the Ad-GFP vs. Ad-Cre-GFP treatment groups and leptin treatment at hypoxic condition; P = 0.006. Data were compared using two-way repeated-measures ANOVA: no leptin vs . leptin vs . leptin + FTY720 within subjects, Ad-Cre-GFP vs . control Ad-GFP viruses between subjects. Sphericity was assessed using Mauchly’s test in both C and D and found not to be violated. All values are expressed as box-and-whisker plots (median, 25%–75% centiles and minimum and maximum values).

Article Snippet: Adenoviral vectors Ad-Cre-GFP containing Cre-recombinase tagged with green fluorescent protein (GFP, 3.3 × 10 12 VP/ml) and control Ad-GFP (3.3 × 10 12 VP/ml) were from Vector Biolabs (Malvern, PA, USA).

Techniques: Knockdown, Activity Assay, Ex Vivo, Transfection, Control, Functional Assay, Whisker Assay

Journal: Scientific Reports

Article Title: Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess

doi: 10.1038/s41598-020-64521-3

Figure Lengend Snippet: Derivation and characterization of cell lines lacking Atp7a , MtI and MtII genes. ( a ) Primary fibroblasts were isolated from the lungs of Atp7a fl/Y ;MtI +/+ /MtII +/+ and Atp7a fl/Y ;MtI −/− /MtII −/− mice and then immortalized by transfection with a plasmid expressing the SV40 large T antigen (SV40 Tag) resulting in WT and MT- cells, respectively. An adenoviral vector encoding CRE recombinase was used to delete Atp7a in WT and MT- cells to obtain ATP7A- and ATP7A-/MT- cells, respectively. ( b ) PCR analysis of genomic DNA was used to confirm deletion of MtI and MtII genes in both the MT- and ATP7A-/MT- cell lines. Expected PCR product sizes: MtI gene (WT = 161 bp; knockout = 176 bp); MtII gene (WT = 282 bp; knockout = 299 bp). ( c ) Immunoblot analysis was used to confirm the loss of ATP7A protein in both ATP7A- and ATP7A-/MT- cell lines. Tubulin was detected as a loading control. Images of full-length gels and immunoblots are provided in the supplementary data.

Article Snippet: To obtain ATP7A- and ATP7A-/MT- cell lines, the floxed Atp7a allele was disrupted in WT and MT- cell lines, respectively, using an adenoviral vector expressing GFP-tagged CRE recombinase (Ad-Cre-GFP 1700; Vector Biolabs).

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Knock-Out, Western Blot, Control