| MAGE-1 gene was firstly reported in melanoma by Van der Bruggen P et al using gene clone technology in 1991. The antigens it coded were found in many kinds of tumor besides melanoma but can't be found in normal tissue except placenta and testis. Further studies shows that MAGE genes are a big family that locate in X chromosome with one ORF(open reading frame). The proteins these genes coded have a same sequence of 200 amino acid. MAGE-A1 gene is about 4.5 kb with 3 extrons. Like many other members, the coding zones all locate in the third extron. Most of present researches focus on the tumor vaccination field because this gene only expressed in malignant tumors and immune privilege sites. Few reports are about the effects of this gene on the tumorigenesis, development and cells growth. In this study, we investigate the effects on NIH3T3 cells through observing the changes of cell cycle, apoptosis and cells mobile ability after transferring the MAGE-A1 gene. Methods1. Extraction of total RNA in BEL7402 cells and amplification of MAGE-A1The total RNA was extracted according to manufacturer's protocol. Using RT-PCR to amplify the MAGE-A1 sequence, the following primers were used for PCR amplification: sense, 5'CCGA ATT CCG GCC ACC ATG GCG ATG TCT CTT GAG CAG AGG AGT-3', antisense, 5' CA CA A GCT TCG GAC TCC CTC TTC CTC CTC TCT -3'. Add EcoR I and Hind III restriction site in both ends respectively.The PCR product was reclaimed from the gel using the reclamation reagent box supplied by TaKaRa, Dalian, China. Synthesis of the primers and measurement of the sequence were completed by TaKaRa, Dalian, China.2. Construction of pEGFP-C3-Al and pcDNA3.1-AlThe plasmid pEGFP and MAGE-A1 sequence were digested with EcoR I and Hind III respectively, recollect the corresponding products, then ligate them to get the reconstructed plasmid pEGFP-C3-Al. The plasmid pcDNA3.1 and MAGE-A1 sequence were digested with EcoR I and Hind III respectively, recollect the corresponding products, then ligate them to get the reconstructed plasmid pcDNA3.1-Al.3. Transfection, screening and observing with fluorescence microscope and laser scanning confocal microscopeAccording to the manufacturer's protocol, we completed the transfection procedure. Firstly, plate 1-3×10~5 NIH3T3 cells in each well of a 6-well plate one day before the transfection experiment. When conducting the experiment, add the plasmids and reconstructed plasmids pEGFP-C3-Al, pcDNA3.1-Al, pEGFP-C3, pcDNA3.1 (each content was 4ug) into the premix of 6 μ 1 FuGENE6 and serum-free medium and incubated at room temperature for 15 minutes to get the total mixture of 200 μ 1. Then, put each mixture into each well respectively. 24 hours later, put G418 into each plate with the content of 500 μ g and changed into 100 μ g one week later. We observed the transfection effects of those transferred plasmids including GFP with fluorescence microscope and laser scanning confocal microscope.4. Immunocytochemical analysisIn order to test the MAGE-A1 protein, we plated the transferred cells into the slide, then tested the expression of MAGE-A1 usingimmunocytochemical peroxidease-conjugated streptavidin (SP) method according to manufacture's protocol. Using BEL-7402 as the positive control and the untransferred cells as the negative control and using the PBS as the blank control.5. Western Blot AnalysisIn order to identify the MAGE-A1 gene expression, we harvested the transferred cells that transferred with plasmid pcDNA3.1 and pcDNA3.1-Al. After cultivating in medium including 1% FBS to homogenize the cells, then perform the Western blot analysis.6. Cells mobile abilityWith 8 um Millicell-PCF, we put 200ul medium into the lower chamber, then put 400ul medium including lxlO5 cells into the upper chamber. After cultured for 8 hours, stained with HE, removed the attached cells on membrane carefully and counted cells that infiltrated the pore in 30 high visions, then we got the number of every 10 high-power microscopic view.7. Cell cycle and apoptosis assayBefore testing, we...
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