Personalized Research Plan for New Tumor Drug Targets
Based on TCGA, GEO big data, or Creative Proteomics database or customer samples, new functional genes that have not been reported to be functional in the tumors that have been studied downstream of the customer's existing research target genes are screened. This experiment verifies and explores the underlying mechanism of the selected genes, and provides preliminary data basis for explaining the mechanism of the target genes.
The whole gene expression profile chip was used to analyze the gene expression changes of the customer's existing research target gene A after interference. Combined with the CP disease key gene patent database and the high-content functional gene screening platform (HCS) to screen out those that have not been reported in the studied tumor Downstream gene B, which plays an important regulatory role in tumor cell proliferation. Complete the functional experiments of the B gene at the cellular and animal level, and continue the research project of the target gene A by supplementing the research method.
Gene chip screening of related genes downstream of target gene A: construction of interfering lentiviruses of target gene A, knockdown of target gene A in tumor cell lines, extraction of total RNA, and full-gene expression profiling chip detection with control group, through informatic analysis , Screen for genes with significant differential expression, select about 25-40 differential genes for qPCR verification, and identify 20 candidate differential genes.
Preliminary screening of target functional genes: 20 candidate gene shRNA lentiviruses were used to infect target cells, and each gene was replicated in 3 wells to reduce the expression of candidate genes endogenously expressed in target cells. After infection, the cells were counted for 4 to 5 days, and the cell proliferation of the cells after the down-regulation of candidate gene expression was compared with the control cells. About 1-2 genes B that had an effect on cell proliferation were initially selected.
Perform a complete cell functional study of one of the functionally positive genes B screened: verify the knockdown efficiency of the target gene B in cells by qPCR and WB, and perform MTT / cell cycle (PI / flow cytometry) on tumor cell lines ) / Apoptotic (Annexin V / Flow cytometry) / BrdU / Clone formation and other proliferation-oriented functional experiments.
Functional study of functionally positive gene B in vivo: Preparation of tumor cells of target gene knockdown experimental group or control, injection of nude mice, breeding of nude mice until tumor growth, recording animal weight, tumor size curve, and post-mortem Collect tumor masses and process the data results.
Study of the mechanism of target gene B: through cellular immunofluorescence, Patharray downstream pathway analysis, downstream classical pathway WB verification, Co-IP mass spectrometry analysis of interacting proteins, dryness detection, aging detection, DNA methylation detection, acetylation detection, pan Supplementary research schemes, such as phytochemical detection and autophagy mechanism testing, verify the possible pathogenic molecular mechanism of target gene B.
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