Fig. 1
Luminescence imaging and genetic validation of HCN4-immunoreactivity in HCN4 conditional knockdown mice. a Schematic diagram representing the genetic constructs of transgenic mice used in this study. From top to bottom: HCN4+/+ (wild-type mouse), HCN4+/tTA_TRE (tetracycline-dependent heterozygous HCN knock-in mouse), HCN4Luc/tTA_TRE DOX(−) (firefly luciferase cDNA and tetracycline-dependent genetic switch double knock-in mouse without doxycycline [DOX] treatment), and HCN4Luc/tTA_TRE DOX(+) (the same double knock-in mouse with DOX treatment). tTA: tetracycline transactivator; TRE-CMV: tetracycline-responsive element fused with a cytomegalovirus minimal promoter. b Ex-vivo luminescence imaging of a transverse lumbar spinal cord section from an HCN4Luc/tTA_TRE mouse. The luminescence intensity is shown as a pseudocolor spectrum. Scale bar, 1 mm. c qPCR analysis of HCN4 mRNA from the transgenic mice shown in a. DOX was administered for 2 weeks. The expression levels of HCN4 relative to 18S rRNA were normalized to that in wild-type mice. * P < 0.03, ** P < 0.01. d-i Transverse spinal sections transgenic mice. d, g HCN4+/tTA_TRE mouse. e, h HCN4Luc/tTA_TRE DOX(−) mouse. f, i HCN4Luc/tTA_TRE DOX(+) mouse. Upper and lower panels are images at low and high magnifications, respectively. In g and h, arrowheads indicate cells showing circumferential immunostaining. Note that both circumferential and punctate immunostainings are not visible in i. Scale bars, 200 μm (d-f) and 50 μm (g- i)