Fig. 2

Generation of INSM1 transgenic model. a A 2.8 kb full length human INSM1 cDNA was cloned into a tetracycline operator expression cassette at pTet-on-Advanced expression vector. The expression cassette consists of seven tetracycline operator repeats (tetO), and a bi-directional CMV mimimal promoter for EGFP and INSM1 expression. The bi-transgenic mice (Bi-Tg) were generated by crossing (tetO)₇-CMV-INSM1 and CCSP-rtTA transgenic mice controlled by 2.3 kb CCSP promoter-linked rtTA sequence and a human growth hormone poly(A) tail. In bi-transgenic mice (CCSP-rtTA x (tetO)₇CMV-INSM1), rtTA is expressed in epithelial cells under the control of CCSP promoter. In the presence of doxycycline (Dox), rtTA binds to TRE element at (tetO)₇CMV promoter and activates the expression of INSM1. b Bi-transgenic mice were fed with Dox food while mating. INSM1 mRNA were assessed at postnatal days 21 (PN21) by Northern blot analysis, c RT-PCR, and d real-time RT-PCR. INSM1 over-expression was detected in the lung tissues of bi-transgenic mice. No INSM1 expression was detected in single transgenic or wild type mice. Without Dox induction, no INSM1 expression in lung was detected. e, f INSM1 expression is detected in Bi-Tg lung and co-localized with CCSP and synaptophysin. No INSM1 signal was detected in wild type or single transgenic mice. g PNEC marker, synaptophysin-positive cells are CCSP-positive in wild type PN7 mouse lung tissue. h At E17.5 lung tissues, INSM1 expression is detected and co-localized with ASCL-1 in wild type and Bi-Tg. Results were represented from at least three mice