Establishing The Transgentic Mouse For A Liver-specific Conditional Cre Recombinase And Lung Cancer Methylome

The transgentic mouse of hepatocyte(liver)-specific and controllable Cre/loxP system is a necessary tool for learning the functions of genes during the development and the tumorgenesis of liver cell in vivo.There have been Albumin promoter and LAP promoter driven Cre-LoxP system in hepatocyte (liver)-specific gene knock-out mouse,but these mice model are still unable to effectively prevent leakage expression of the target gene and the embryonic lethality in occasion that the knocked gene is important to the development of mice.Here we try to use hepatocyte(liver)-specific promoter driven Tet-Off system to establish an efficient hepatocyte (liver)-specific and condition-specific, tamoxifen-mediated Cre-ERT2 transgenic mouse.Among 15 founder strains by this dual-controllable constructs into the mouse' ES cells.,The cross with the Loxp-LacZ (ROSA26)reporter mouse strain for the offsprings has been carried out and checking by RT-PCR and LacZ staining for the inducible expression of the Cre by both Dox and Tamoxifen.We have failed to find hepatocyte(liver)-specific strain,except for two kidney-specific mouse strains with Cre-ERT2 expression.In order to obtained the hepatocyte(liver)-specific conditionally regulated Cre-ERT2 mouse strains,we have already started to replace LAP promoter with the albumin promoter/enhancer in the system for the new round of effort. Lung cancer is one of the most common malignant tumor with very high incidence and mortality in the forefront of disease spectrum,and the incidence is increasing year by year.The five-years survival rate of lung cancer is 15%, and the annual number of deaths from lung cancer is more than 1 million.Early diagnosis of lung cancer patients is the key determinant for better therapeutic outcome.Although imaging and cell screening strategy has been used for many years,inadequante sensitivity and specificity of which results are in little improvement of the clinical treatment. In addition to the genetic defects,cancer suffers from the epigenetic abnormality extensively. DNA methylation is the most well characterized epigenetic entity, its utility in cancer diagnosis has been increasingly appreciated.We have used the MBD (methylated DNA binding domain) mediated affinity chromatography(Methyl-capture) to fractionate the DNA fraction according to the density of the CpG methylation.The DNA fraction enriched with approaximately 21 CpG methylated DNA from both normal lung tissue and a mixed population of lung cancer cell lines was sequenced by Solexa (Illumina).We have confirmed the methylated state of several informative candidates that have been suggested from the methylomic approach by bisulfite-sequencing PCR and methylation-specific PCR.This study has demonstrated the merit of our platform technology for the methylome of the mammanlian cells from both throughtput and precision perspective.Finally, the list of the candidates identified from this process provides plenty of information for the efforts to the mechanistic elucidation of lung cancer genesisi and development of the DNA methylation based cancer diagnostics.