Capture Of Transcriptional Active DNA Fragments In Metastatic Mouse Breast Cancer Cells

Breast cancer is one of high incidence cancer and mortality of malignant tumors in the world among women. With the completion of human genome sequencing, especially the discovery of non coding RNA in the genome in recent years, these results suggest that, a large number of gene associated with life in genome especially disease related, yet to be discovered and studied.Therefore, this study expect to discover new cancer related genes by promoter trap. With the metastatic breast cancer cells as model, genomic DNA random fragments were inserted into reporter gene vector, the transcriptional activity of the DNA fragments were detected by reporter gene activity, thus DNA fragments with transcriptional activity were obtained to establish the basis for further determination of the corresponding genes.Contents and the main study results are as follow:1. Construction random plasmid library of mouse 4T1 breast cancer cells genomic DNA and promoterless vector pCpGfree-basic,706 plasmids were acquired and construct secreted alkaline phosphatase (SEAP) positive control vector;2.706 plasmids were transfected into 4T1 cells culured in 24-well culture plat, and the supernatant was collected after transfection of 36h. The expression of the reporter gene SEAP was detected, and the positive plasmids were identified.;3. The positive plasmids were sequenced, and the sequences were obtained. The Sequence alignment analysis was performed to determine whether the specific location of the chromosome and the genes in the upper and lower reaches were regulated.According to the results,15 positive plasmids whose sequence with transcriptional activity, cbfa2t3 gene promoter, and seven in different gene intron and the rest is located in DNA non coding area. This provides the basis and reference for the follow-up study of the genes related to breast cancer.