| Objective: Currently the malignant tumor is one of diseases which endanger seriously the human life and health in the world. The study of the tumorigenic mechanism and the development of the anti-tumor drug suffer the worldwide concern. In the discussion of the tumorigenic mechanism and the screening of anti-tumor drugs, people often use the animal model of transplanted tumor. To compare the effect of different drugs, the animal models of transplanted tumors built should be stable. If want to keep the stability of the animal models of transplanted tumors, we have to give quality control on the tumor cell strains. Scientific research and drug screening demand the quality-controlled tumor animal model, but the domestic and foreign research on this hand is still inadequate. Presently there are about 500 kinds of transplanted tumors collected in the world, most of which belong to the mouse tumor. Sarcoma 180(S180) is one of the most usually used mice transplanted tumor cell lines and widely applied to copy the mouse tumor animal models. This cell line has been set up for nearly 100 years and introduced into the domestic for 50 years. Many scholars reported it has already undergone biggish genetic difference and the cell strains conserved in different institutes have also had very large difference. Wang xiufang etc (2000) made S180 cloning and got 5 clonal cell strains, including S180-S2D9,S180-S1B11,S180-S1F11,S180-S1H10,S180-S2D7. Herein S180-S2D9's character is homogeneous and basically keep original S180'character, which had been collected by Chine Center for Type Culture Collection. Wang xiufang etc have already chose the known single antibody and nucleotide probe to react with S180-S2D9, primarily setting up check ways to find the genetic variance of the tumor cell strain. Modern molecular biology technique may directly analyze DNA of tumor cell strains and it brings the quality control of tumor cell strains to a new field. Random amplified polymorphic DNA(RAPD) adapts to biological research of any an unknown gene nucleotide sequence and can objectively reflect whole genomic character. In recent years there are numerous reports about research of the diversity of species heredity with RAPD. This study is planned to pick out the specific primers from 23 primers, which are able to distinguish 5 clonal cell strains from S180. We expect it can render the acessibility to analysis and research RAPD character of 5 S180 clonal cell strains, and S180-S2D9 passage cells' character from cell karyological characteristics,DNA contents and mice life span, furtherly to provide basic material for comparament and analysis of genetic background. Method: Firstly, analysis and research of RAPD character of 5 S180 clonal cell strains(S180-S2D9,S180-S1B11,S180-S1F11,S180-S1H10,S180-S2D7) by employing 23 primers: 1. Preparation of genomic DNA. Gathering passage cells amount at 1×108, washing them with PBS liquid and resuspended in TE buffer liquid , adding DNA extraction buffer , mixing and incubated in water bath at 55°C overnight, and phenol equilibrited with Tris-Cl and chloroformï¼isoamyl alcohol(24:1) extracting respectively, adding NaAC and anhydrous ethanol to deposit the genomic DNA, rinsing pellets with 75% ethanol twice and air drying, resuspending pellets in sterile ddH2O and used later. 2.Analyzing RAPD character: The genomic DNA of 5 clonal strains from S180 including S180ï¼S2D9,S180ï¼S2D7,S180ï¼S1F11,S180ï¼S1H10,S180ï¼S1B11 are amplified by using RAPD-PCR with 23 single primers. The reaction program includes 40 cycles. Every cycle involves denaturation for 1min at 94℃ ,annealing for 1 min at 36℃ and extention for 2 min at 72℃. Extention for 10 min at 72℃after the last cycle and denaturation for 2 min at 94℃ before the first cycle. The amplification products are observed with electrophoresis on agarose gel. Secondly, after extracting genomic DNA, the first generation,the 25th generation,the 50th generation and the 75th generation cells of S180-S2D9 are analyzed by RAPD with the...
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