Development Of A Transgenic Reporter Mouse Model Expressing Both EGFR^L858R And Luciferase

Epidermal growth factor receptor(EGFR), a member of ErbB family, is an important transmembrane receptor with signal-transducing tyrosine kinase activity. It was reported that mutations in the tyrosine kinase domain of the epidermal growth factor receptor gene had been found in a subset of patients with lung cancer. And they were sensitive to EGFR tyrosine kinase inhibitors. Some patients with non-small-cell lung cancer have specific mutations on EGFR gene, which was relating with clinical responsiveness to the tyrosine kinase inhibitor Gefitinib. Thus, EGFR tyrosine kinase inhibitor achieves more and more attention because of its importance on the therapy of non-small cell lung cancer(NSCLC).The purpose of this study is to construct several familiar EGFR mutations in patients with non-small-cell lung cancer who have clinical responsiveness to the tyrosine kinase inhibitor, and to develop a transgenic reporter mouse model expressing both luciferase and one of the mutations EGFRL858R. Thereby, tumours can be observed by in vivo bioluminescence imaging technique in this transgenic reporter mouse model, which provides a convenient tool for the research of EGFR targeted therapy of non-small-cell lung cancer.Tet-On Advanced System is an inducible eukaryotic expression system. In this system, expression is activated by the addition of doxycycline (Dox), a tetracycline(Tc) deriviative. As a result, gene expression will be tightly regulated in response to varying concentrations of Dox. The regulation of Tet Systems, mainly on the genes of tetracycline-resistance operon of Tn10 transposon by the Tet repressor protein (TetR), is highly specific and stable. TetR blocks transcription of these genes by binding to the tet operator sequences (tetO) in the absence of Tc. In contrast, TetR dissociates from tetO and transcription of resistance-mediating genes begins.In present study, surfactant protein A promoter (SP-A promoter) is used to regulate the expression of the reverse tet-controlled transcriptional activator(rtTA), which will result in the expression of mutant EGFR gene in lung of transgenic mice. Murine SP-A promoter, amplified by polymerase chain reaction(PCR) from mouse genomic DNA, was cloned into pGEM-T vector and sequenced. For a pSP-rtTA vector, PCMV of pTet-On vector was replaced with SP-A promoter. And the full-length EGFR cDNA was acquired by overlapping PCR from human lung adenocarcinoma A549 cells. The EGFR mutations, including L858R, delE746-A750 and delE746-A750/T790M, were obtained by point Mutagenesis and cloned into pTRE-Tight-BI-L vector separately, which would come to vectors pTRE-EL-1, pTRE-EL-2 and pTRE-EL-3.To obtain a stable SP-rtTA cell line expressing rtTA, human small cell lung cancer cell line NCI-H446 was transfected with pSP-rtTA vector, and selected with G418. After transfected with pTRE-Tight-BI-L vector and induced by doxycycline, the fluorescence of the twenty-one SP-rtTA cell clones caused by the expression of the luciferase gene was assessed to confirm the expression of rtTA protein. Following that, RT-PCR method was utilized to evaluate SP-rtTA cell clones obtained. For a double stable H446-rtTA-EL-1 cell line which can express both EGFR and Luciferase, the stable SP-rtTA cell line was transfected with pTRE-EL-1 vector, and selected with hygromycin. Also RT-PCR method was used to check the thirteen H446-rtTA-EL-1 cell clones induced by Dox. The activity of the fluorescence was assayed through adding different concentrations of Dox into the H446-rtTA-EL-1 cell line. What's more, the expression of EGFR was evaluated by western blot. It was showed that the activitv of the fluorescence and the expression of EGFR increased with the increasing concentration of Dox gradually, which suggested the Tet-On system we developed was effective and could be used for in vivo research.In this study, Plasmid pSP-rtTA was digested by Hindâ…¢, purified and microinjected into male nuclei to develop transgenic mouse model. After the transplantation of 465 ova into 17 pseudopregnant mice, 121 offspring were obtained, of which 15 (9 males and 6 females) were positive, it came to a rate of 12.40%. Meanwhile, plasmid pTRE-EL-1 digested by Scaâ… was injected into male nuclei to generate transgenic mouse model. 94 offspring were obtained after 360 ova were transplanted into 12 pseudopregnant mice, of which 6 were positive, it achieved a rate of 6.38%. Analysis revealed that the rtTA was expressed only in lung, and no expression was found in other tissues or organs, such as brain, stomach, heart, liver, spleen or kidney. The two transgenic mice lines were developed for a large number by mating with each other. After induced by Dox, the offspring were evaluated, including the activity of the fluorescence and EGFR, to obtain the positive ones we need.