A Stringent Dual Control System Overseeing Transcription And Activity Of The Cre Recombinase For A Robust Liver-specific Conditional Gene Knock-out Mouse Model

The most stringent approach to study the gene function in vivo in development is its knock-out in both spatial(cell type specific) and temporal (development stage specific) manners,which is commonly achieved by use of a genetic modified mouse strain with its Cre recombinase activity under the control of the needed precision and pattern.Cre recombinase-mediated site-specific recombination of chromosomally integrated loxP sites allows us to create defined gene deletions or mutations in many kinds of tissues in mice thus provides unprecedented possibilities for the functional studies of the mammalian genome and for the generation of animal models for human diseases.Hepatocellular carcinoma is one of the most devastating types of human cancer in our country.The undesirable expression (leaky) of transgene and the embryonic lethality result in the absentee of a hepatocyte(liver)-specific Cre mouse model which has hampered the efforts to understand of the causes of this disease,as well as the development of the better measures to confine the hepatocellular carcinoma.In our pursuit to establish the mouse model equipped with the hepatocyte (liver)-specific regulable Cre recombinase gene,a dual mechanistic approach has been taken:First,the trans-acting factor in Tet-off regulable system is driven by the promoter of the CCAAT/enhancer binding proteinβ(C/EBPβgene,a well established hepatocyte specific gene) and second,the tamoxifen-mediated functional control(the estrogen receptor mediated) at the protein-protein interaction level.The critical assessment in cell culture system has shown the desirable profile of both hepatocyte-specificity and regulability of the Cre expression.So,transgenic mice expressing such a hepatocyte(liver)-specific regulable Cre recombinase gene may provide a irreplaceable and useful genetic tool to create liver-specific knock-out mouse and mouse model for hepatocellular carcinoma. Oncolytic adenoviruses,which replicate selectively in cancer cells and therefore result in cancer-specific cytotoxicity is a promising approach to cancer treatment by the genetic intervention.However,no successful story has yet to be seen in the clinical trials.This unsatisfactory status has attributed to the inadequancy in the potency and specificity of the current system to the cancer cells versus their normal counterparts.The tumor suppressor protein p53 is a transcription factor that can positively regulate the expression of critical target genes involved in prohibiting cell growth or induction of apoptosis; p53 is also able to suppress the transcription of other genes by binding to components of the basal transcription elements.We have exploited the loss of p53 function in the regulation of gene transcription to develop a novel gene therapy vector that maximizes the expression of the potential therapeutic gene in tumors while simultaneously down-regulates the same gene in normal cells. First,the potential therapeutic gene(or a luciferase reporter gene) is placed under the control of a promoter with high activity perticularly in tumor cells and can be repressed by wtp53.Residual expression of the target gene in normal cells is then repressed by an anti-transcription of the target gene by a reversely orientated promoter which can be activated by wtp53.The expected p53 dependent profile of this new form of the dual control system has been demonstrated first in a transit transfection/luciferase reporter assay and then with the E1A gene of the adenovirus.The oncolytic adenoviruses that under production will be tested in the tumor animal models for its untility in the future anticancer practice.