The STZ-treated group (n = 4). doi:10.1371/journal.pone.0060411.gIns1-luc BAC Transgenic

The STZ-treated group (n = 4). doi:10.1371/journal.pone.0060411.gIns1-luc BAC Transgenic

The STZ-treated group (n = 4). doi:10.1371/journal.pone.0060411.gIns1-luc BAC Transgenic MiceFigure 5. Bioluminescence images of Ins1-luc BAC transgenic mice fed a regular (RD) or a high-fat diet (HFD). (A) Representative images of mice fed a RD or an HFD for 12 weeks beginning at 6 weeks of age. (B) Quantification of bioluminescence intensity (RD: n = 4; HFD: n = 6). *P = 0.019. (C) Computed tomography (CT)-based body composition analysis. Representative CT images of mice treated with a RD or an HFD for 8 weeks. Purple, yellow, and blue areas represent visceral fat, subcutaneous fat, and lean mass, 22948146 respectively. (D) Body fat percentage of mice treated with a RD 12926553 (n = 4) or an HFD (n = 5) for 8 weeks. (E) Fasting blood glucose levels in the RD- (n = 4) or HFD- (n = 5) treated groups of mice for 8 weeks. (F) b-cell mass in the RD- (n = 4) and in the STZ- (n = 5) treated group. P = 0.49 (G) Immunohistochemistry for anti-insulin (Ins) and anti-luciferae (Luc) antibodies with diamidino-2-phenylindole (DAPI) for nuclear staining in the islets of RD- and HFD-fed Ins1-luc BAC transgenic mice Scale bars: 50 mm. doi:10.1371/journal.pone.0060411.gHistological analysisWT and Ins1-luc BAC transgenic mice were euthanized at 8 weeks of age, and the pancreatic tissues removed. Tissues were fixed in 10 formalin and embedded in paraffin. Tissue sections were incubated with guinea pig anti-insulin antibody (Abcam, Cambridge, UK) and rabbit anti-glucagon antibody (Linco Research, St. Charles, MO, USA) for 8 hours at 4uC. The antigens were visualized using appropriate secondary antibodies conjugated with alexa488 and alexa594 with nuclear staining using diamidino-2phenylindole (DAPI) (Invitrogen). For measurement of b-cell mass,the removed pancreatic fragments were immediately weighed. Ten consecutive 5-mm sections 200 mm apart spanning the entire pancreas were stained with anti-insulin antibody. Images of the sections were scanned and analyzed using a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan) and BZ-II AKT inhibitor 2 analyzer software (Keyence). The relative areas stained for insulin were measured and multiplied by the pancreas weight to estimate the b-cell mass.Ins1-luc BAC Transgenic Micevector. To create expression clones and produce recombinant adenoviruses, the pENTR4 inserts were transferred into the pAd/ CMV/V5-DEST destination vector using the LR recombination reaction, and PacI-linearized plasmids were transfected into 293A cells (Invitrogen) using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland). MedChemExpress Gracillin Adenoviral constructs (5.06109 pfu/mouse) were injected via the tail vein, and serial BLI was performed before and after infection on designated days. On day 0, the initial BLI was performed before the viral infection.Statistical analysisData were expressed as the means 6 standard errors of the means and compared using an unpaired t test. Probability values of less than 0.05 were considered significant.ResultsWe first obtained 68 founder (F0) mice. Of those 68 mice, 8 were transgenic, as confirmed by luciferase gene integration identified by PCR. On BLI screening of the 8 F0 transgenic mice, 4 mice did not emit any BLI signal, 2 mice exhibited an ectopic bioluminescence signal, and the remaining 2 mice exhibited considerable signal intensities emanating from the pancreatic region. Finally, germ line transmission was confirmed by transgene-specific PCR in 1 of the remaining 2 mice (Ins1-luc BAC transgenic mouse). For further experiments,.The STZ-treated group (n = 4). doi:10.1371/journal.pone.0060411.gIns1-luc BAC Transgenic MiceFigure 5. Bioluminescence images of Ins1-luc BAC transgenic mice fed a regular (RD) or a high-fat diet (HFD). (A) Representative images of mice fed a RD or an HFD for 12 weeks beginning at 6 weeks of age. (B) Quantification of bioluminescence intensity (RD: n = 4; HFD: n = 6). *P = 0.019. (C) Computed tomography (CT)-based body composition analysis. Representative CT images of mice treated with a RD or an HFD for 8 weeks. Purple, yellow, and blue areas represent visceral fat, subcutaneous fat, and lean mass, 22948146 respectively. (D) Body fat percentage of mice treated with a RD 12926553 (n = 4) or an HFD (n = 5) for 8 weeks. (E) Fasting blood glucose levels in the RD- (n = 4) or HFD- (n = 5) treated groups of mice for 8 weeks. (F) b-cell mass in the RD- (n = 4) and in the STZ- (n = 5) treated group. P = 0.49 (G) Immunohistochemistry for anti-insulin (Ins) and anti-luciferae (Luc) antibodies with diamidino-2-phenylindole (DAPI) for nuclear staining in the islets of RD- and HFD-fed Ins1-luc BAC transgenic mice Scale bars: 50 mm. doi:10.1371/journal.pone.0060411.gHistological analysisWT and Ins1-luc BAC transgenic mice were euthanized at 8 weeks of age, and the pancreatic tissues removed. Tissues were fixed in 10 formalin and embedded in paraffin. Tissue sections were incubated with guinea pig anti-insulin antibody (Abcam, Cambridge, UK) and rabbit anti-glucagon antibody (Linco Research, St. Charles, MO, USA) for 8 hours at 4uC. The antigens were visualized using appropriate secondary antibodies conjugated with alexa488 and alexa594 with nuclear staining using diamidino-2phenylindole (DAPI) (Invitrogen). For measurement of b-cell mass,the removed pancreatic fragments were immediately weighed. Ten consecutive 5-mm sections 200 mm apart spanning the entire pancreas were stained with anti-insulin antibody. Images of the sections were scanned and analyzed using a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan) and BZ-II analyzer software (Keyence). The relative areas stained for insulin were measured and multiplied by the pancreas weight to estimate the b-cell mass.Ins1-luc BAC Transgenic Micevector. To create expression clones and produce recombinant adenoviruses, the pENTR4 inserts were transferred into the pAd/ CMV/V5-DEST destination vector using the LR recombination reaction, and PacI-linearized plasmids were transfected into 293A cells (Invitrogen) using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland). Adenoviral constructs (5.06109 pfu/mouse) were injected via the tail vein, and serial BLI was performed before and after infection on designated days. On day 0, the initial BLI was performed before the viral infection.Statistical analysisData were expressed as the means 6 standard errors of the means and compared using an unpaired t test. Probability values of less than 0.05 were considered significant.ResultsWe first obtained 68 founder (F0) mice. Of those 68 mice, 8 were transgenic, as confirmed by luciferase gene integration identified by PCR. On BLI screening of the 8 F0 transgenic mice, 4 mice did not emit any BLI signal, 2 mice exhibited an ectopic bioluminescence signal, and the remaining 2 mice exhibited considerable signal intensities emanating from the pancreatic region. Finally, germ line transmission was confirmed by transgene-specific PCR in 1 of the remaining 2 mice (Ins1-luc BAC transgenic mouse). For further experiments,.

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