The Generation Of DAAO Gene And YCD Gene Transgenic Mice Carrying A Reporter Gene

With the development of molecular biology and the correlated discipline, gene therapy of tumor has attract more and more attention, and suicide gene therapy may be used as a promising method for treating tumor.To study the applicable prospect of suicide gene on tumor therapy in clinic,we cloned the gene of D-amino acid oxidase (DAAO) and the gene of yeast cytosine deaminase(YCD).We hope to establish the transgenic mices of DAAO gene and YCD gene,and the two transgenic strain mices are valuable animal system for studying the biological characteristic of DAAO gene and YCD gene ,for studing the killing activity of DAAO/D-Ala and YCD/5-FC gene therapy systems on tumor.The D-amino acid oxidase gene derived from R.gracilis and it could oxidize D-amino acids(D-Ala). Hydrogen peroxide(H2O2) is a reactive oxygen species(ROS) generated in the deamination of D-Ala catalyzed by DAAO. H2O2 readily crosses cellular membranes and yields hydroxy free radical by oxidation or deoxidation,which could kill cells by damaging DNA, proteins, and lipids. DAAO has a low level activity in mammal cell and it is only found at corpuscles peroxide ,moreover D-Ala is not found in mammal animal, so DAAO can be used as a new safe suicide gene in gene therapy of tumor. Bacterial cytosine deaminase(BCD) is a enzyme lied in bacteria. This enzyme activity is only found in prokaryotes. BCD is able to convert the nontoxic 5-FC into 5-FU, and 5-FU is a abroad applying chemotherapy drug which could kill most tumor cells by disrupting DNA synthesis.In the current study, the yeast CD proved superior to the bacterial CD at eliminating tumors in vivo because of its ability to convert 5-FC into 5-FU more efficiently,so the system of YCD/5-FC is more efficient than the system of BCD/5-FC at the treatment of tumors.In the present study, we constructed DAAO gene expression vector plRES-DAAO using the enhanced green fluorescent protein(EGFP) gene as a reporter gene, which contained CMV promoter and DAAO gene open reading frame by recombination DNA technique. We also constructed YCD gene expression vector pIRES-YCD using the red fluorescent protein(RFP) gene as a reporter gene, which contained CMV promoter andYCD gene open reading frame.They were identified by restriction endonucleases digestion. In vitro, the Hela cells were transfected with plRES-DAAO plasmids and pIRES-YCD plasmids, EGFP and RFP were seen in the transfected cells under fluorescent microscope after 48 hours.The transfected cells were treated with D-Ala or 5-FC and MTT colorimetric assay was used to detect the transfected cells' activity. The result showed that D-Ala and 5-FC could kill respectively DAAO or YCD transgenic cells.These results proved that DAAO gene and YCD gene had a good active expression. After restriction enzyme BglII digestion, the coding elements of pIRES-DAAO were microinjected into male pronuclei of mice zygotes. Following microinjection, the embyros were transferred to oviducts of psedopregnant females. 19 pups were born and survived. 4 of them were verified to integrate DAAO gene and EGFP gene in their genomic DNA by multiplex PCR assay, named C57-TgN (DAAO) SMMU. At the same time.we acquired a abnormally dead young mice, which was verified to integrate the DAAO gene and EGFP gene in its genomic DNA by multiplex PCR assay. Cryosection was fabricated and EGFP was seen in most part of constitution under confocal laser scanning microscope. After restriction enzyme BstBl digestion, the coding elements of pIRES-YCD were microinjected into male pronuclei of mice zygotes at the same method. 9 pups were born and survived. 2 of them were verified to integrate YCD gene and RFP gene in their genomic DNA by multiplex PCR assay, named C57-TgN (YCD) SMMU.The microinjection work is still going on.In the present study, we also studied the relationship between DAAO/D-Ala system and apoptosis in vitro,so did the relationship between YCD/5-FC system and apoptosis.The Hela cells, transfected with pIRES-DAAO plasmids and pIRES-YCD plasmids, resulted in typical apoptosis phenom...