Human Carcinoma Of Esophageal Eca9706 Cell DNA Polymerseβ Gene Targeting

Background and Objective:The interaction between environmental factors and hostal factors lead to the development of carcinoma. The cells' sensitivity to blastomogen and repairing ability to selfmutation have directed effects to the developed probability of canceration. DNA polymerseβ(polβ) is a housing keeping gene, belonging to the eukaryocyte DNA polymerse family.It is one of the most important repairing enzyme and it's main function is base excision repair. From fermentum to mammal cells, DNA polβis highly conservative, widely existed in mammal cells. polβis 35kb, located in the 8th chromosome and near to the centromere. It is a single copied and split gene, containing 14 extrons and 13 introns. Its expression product is a single strand micromolecule protein, relative molecular weight is 39KD. It's main function is concerned as DNA damage repairing, especially in the course of base excision repairing, it can make up nucleotide gaps.Since the whole cDNA of DNA polymerseβwas cloned by DNA recombination technology, people begin to pay more attention to its biofunction. Recent years, many hakeems do systematical researches about DNA polymerseβin tumour, mutational cell and its expressional condition. These researches offer a new stage to demonstrate the molecular mechanism of carcinoma development.At present, the research which aim directly at human DNA polβis more and more deeply, transfecting wild-type polβto elevate normal expression and siRNA to depress mutational and wild-type polβexpression or looking for some other strongly aimed polβbio-moderator to regulate and retrieve the expression of abnormal or mutational DNA polβ. But, the key words to restrict the aim is we don't have DNApolβgene targeting cell line, in the non-gene targeting cell there will have defferent levels of background cell influences, the experimental data and consequence cannot reflect the objective biological effect. This issue have restricted DNA polβfurther bascial study, eventually will be the neck to prevent and treat tumor.Accordingly, this topic introduce gene targeting technology to construct esophageal carcinoma DNA polymerseβgene targeting vector, then introducting the vector into Eca9706, screening and cultivating the cell, identifying the expression of polβmRNA and the existence of DNA gene targeting segment, eventually, construct the model of esophageal carcinoma Eca9706 cell,it lays a solid experimental foundation to study DNA polβbio-function. Methods:According to GenBank , retrieving polβsequence, using Blat (offered by UCSC) to analyze polβextron and intron; choose the targeted region of polβup and down homologous sequence; design and synthesize the homologous recombination primer of up and down sequence; extract esophageal carcinoma Eca9706 cell genome DNA; using PCR to amplify UP and DOWN homology sequence; then cloning into T-vector; screening and identifying the recombinations pGEM-T-UP and pGEM-T-DOWN by a-complement and PCR; analyzing the inserted sequence; using XhoI / Apa I concis pGEM-T-UP and using Bgl II/Hind III concis pGEM-T-DOWN and pcDNA3.1; then subcloning DOWN and UP fragments into frame vector pcDNA3.1, then get the gene targeting vector pOUT-down-up; concising and linearizing the vector by EcoRV; introducting it into Eca9706; Screening positive cell by G-418; at last, using PCR to identify if the DNA gene targeting region have been knocked out and using RT-PCR to identify the expression of mRNA.Results:1. Ampilfied Eca9706 cell genome DNA fregments by PCR; then obtained UP and DOWN homologous sequences, we can see two bright straps in the electrophoresis locate in 1268bp and 1756bp, conforming to the design.2. Conjuncted the aimed genes with T-vector, then transform into DH5a, screening to identify the positive recon of pGEM-T-UP and pGEM-T-DOWN. Sequencing the inverted sequences, conforming to the design.3. Subcloning to obtain gene targeting recon pOUT-down-up, using PCR to identify the recon, conforming to the design.4. linearing the recon by EcoRV, then introducting into Eca9706 cell, screening by G-418 about 6 weeks, at last, obtained the positive Eca9706 cell containing neo gene.5. PCR identified that polβgene targeted regions have been knockout; RT-PCR can not detect polβmRNA.Conclusions:1. Constructed the gene targeting recon pOUT-down-up aimed directly to polβsuccessfully.2. Screened and obtained esophageal carcinoma DNA polβgene targeting cell, offered an ideal cyto-model for the further research of polβbiofunction.