The pups from Ins1-luc BAC transgenic mice and WT mice were Apocynin identified by transgene-specific PCR (Figure 1B). To examine which organs expressed the reporter gene, different organ tissues at 6 weeks of age were dissected, and the luciferase activities determined. Luciferase activity was detected in the pancreatic extracts (3.562.46104 RLU/mg protein; n = 3), but not in the other tissue extracts including those from the thymus and pituitary glands, indicating that the reporter gene was expressed only in the pancreas (Figure 1C). Further immunohistochemical analysis using anti-insulin and anti-luciferase antibodies demonstrated that luciferase-expressing cells were 12926553 colocalized only with the insulinpositive cells in the pancreas; however, only 11.965.2 (n 
= 3) of the insulin-expressing cells expressed luciferase (Figure 1D). Treatment of islets isolated from Ins1-Luc BAC transgenic mice with a proteasome inhibitor, MG132, resulted in coexpression of luciferase by most of the insulin-positive cells, suggesting that the relatively low frequency of the expression of the reporter in b cells depends mostly on the proteasomal degradation of luciferase (Figure S1). Using quantitative PCR analyses of genomic DNA isolated from MIP-Luc-VU and Ins1-luc BAC transgenic mice, the transgene copy number of the Ins1-luc BAC transgenic mice was 374913-63-0 manufacturer determined to be 3, the same as that of the MIP-Luc-VU mice (data not shown) [8]. Quantitation of bioluminescence in vivo critically depends on substrate availability and light-emission kinetics. To determine the kinetics of bioluminescence, MIP-Luc-VU and Ins1-luc BAC transgenic mice were imaged 2.5, 5, 10, 15, and 30 minutes after luciferin injection (5 mg/kg body weight, IP). Consistent with previous findings, levels of bioluminescence in MIP-Luc-VU mice peaked at approximately 10 minutes [8], whereas in Ins1-luc BAC transgenic mice, they peaked at 5 minutes (Figure 2A, B). Comparative measurements of the peak BLI signals of the pancreatic regions at 6 weeks of age revealed that the intensity in the Ins1-luc BAC transgenic mice (2.760.846106 photons/sec; n = 18) was significantly enhanced, by approximately 4-fold that of the MIP-Luc-VU mice (5.660.816105 photons/sec; n = 6; P = 0.022) (Figure 2C, D). As shown by laparotomy, the luminescence of the Ins1-luc BAC transgenic mice emanated onlyFigure 6. Bioluminescence images of Ins1-luc BAC transgenic mice with combined Pdx1, NeuroD, and MafA gene transfer in the liver. (A) Representative images of mice before (day 0) and after the gene transfer. Designated days indicate days after the infection. Each image is optimally adjusted using Living Image software because a huge difference in luminescence from the pancreas and the liver disables showing images with the same longitudinal photon scale. (B) Bioluminescence images of extracted organs from Ins1-luc BAC transgenic mice 3 days after infection. L, P, and S indicate the liver, pancreas, and spleen, respectively. (C) Immunohistochemistry for antiinsulin antibody of the liver from a WT mouse 3 days after infection. Scale bar: 50 mm. Arrows indicate insulin-positive cells. doi:10.1371/journal.pone.0060411.gDiabetes inductionIns1-luc BAC transgenic mice were rendered diabetic at 6 weeks of age by an IP injection of streptozotocin (STZ, Sigma) at a dose of 200 mg/kg body weight in 0.1 M citrate buffer (pH 4.5).High-fat DietIns1-luc BAC transgenic mice were fed either a control regular diet (RD) or a high-fat diet (HFD) con.The pups from Ins1-luc BAC transgenic mice and WT mice were identified by transgene-specific PCR (Figure 1B). To examine which organs expressed the reporter gene, different organ tissues at 6 weeks of age were dissected, and the luciferase activities determined. Luciferase activity was detected in the pancreatic extracts (3.562.46104 RLU/mg protein; n = 3), but not in the other tissue extracts including those from the thymus and pituitary glands, indicating that the reporter gene was expressed only in the pancreas (Figure 1C). Further immunohistochemical analysis using anti-insulin and anti-luciferase antibodies demonstrated that luciferase-expressing cells were 12926553 colocalized only with the insulinpositive cells in the pancreas; however, only 11.965.2 (n = 3) of the insulin-expressing cells expressed luciferase (Figure 1D). Treatment of islets isolated from Ins1-Luc BAC transgenic mice with a proteasome inhibitor, MG132, resulted in coexpression of luciferase by most of the insulin-positive cells, suggesting that the relatively low frequency of the expression of the reporter in b cells depends mostly on the proteasomal degradation of luciferase (Figure S1). Using quantitative PCR analyses of genomic DNA isolated from MIP-Luc-VU and Ins1-luc BAC transgenic mice, the transgene copy number of the Ins1-luc BAC transgenic mice was determined to be 3, the same as that of the MIP-Luc-VU mice (data not shown) [8]. Quantitation of bioluminescence in vivo critically depends on substrate availability and light-emission kinetics. To determine the kinetics of bioluminescence, MIP-Luc-VU and Ins1-luc BAC transgenic mice were imaged 2.5, 5, 10, 15, and 30 minutes after luciferin injection (5 mg/kg body weight, IP). Consistent with previous findings, levels of bioluminescence in MIP-Luc-VU mice peaked at approximately 10 minutes [8], whereas in Ins1-luc BAC transgenic mice, they peaked at 5 minutes (Figure 2A, B). Comparative measurements of the peak BLI signals of the pancreatic regions at 6 weeks of age revealed that the intensity in the Ins1-luc BAC transgenic mice (2.760.846106 photons/sec; n = 18) was significantly enhanced, by approximately 4-fold that of the MIP-Luc-VU mice (5.660.816105 photons/sec; n = 6; P = 0.022) (Figure 2C, D). As shown by laparotomy, the luminescence of the Ins1-luc BAC transgenic mice emanated onlyFigure 6. Bioluminescence images of Ins1-luc BAC transgenic mice with combined Pdx1, NeuroD, and MafA gene transfer in the liver. (A) Representative images of mice before (day 0) and after the gene transfer. Designated days indicate days after the infection. Each image is optimally adjusted using Living Image software because a huge difference in luminescence from the pancreas and the liver disables showing images with the same longitudinal photon scale. (B) Bioluminescence images of extracted organs from Ins1-luc BAC transgenic mice 3 days after infection. L, P, and 
S indicate the liver, pancreas, and spleen, respectively. (C) Immunohistochemistry for antiinsulin antibody of the liver from a WT mouse 3 days after infection. Scale bar: 50 mm. Arrows indicate insulin-positive cells. doi:10.1371/journal.pone.0060411.gDiabetes inductionIns1-luc BAC transgenic mice were rendered diabetic at 6 weeks of age by an IP injection of streptozotocin (STZ, Sigma) at a dose of 200 mg/kg body weight in 0.1 M citrate buffer (pH 4.5).High-fat DietIns1-luc BAC transgenic mice were fed either a control regular diet (RD) or a high-fat diet (HFD) con.
