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In silico Analysis and Site-directed Mutagenesis of Promoters

启动子的计算机模拟分析和定点诱变

Salah Boudjadi Salah Boudjadi
Jean-Francois Beaulieu Jean-Francois Beaulieu

发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2181 浏览次数: 10960

评审: HongLok LungRaghuveer KavarthapuAnonymous reviewer(s)

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Abstract

In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification of these response elements is important to characterize targets of transcription factors in order to understand their contribution to gene regulation. Here, we describe In silico analysis coupled to selected mutagenesis and promoter gene reporter assay procedures to identify and analyze response elements in the proximal promoter sequence of genes.

Keywords: Site-directed mutagenesis (定点诱变) search in silico analysis (计算机模拟分析) search Gene expression (基因表达) search Promoter gene-reporter assay (启动子基因 - 报告基因测定) search Response elements (响应元素) search Transcription factors (转录因子) search

Background

The impact of a transcription factor on global gene expression can be studied through its knockdown using shRNA or CRISPR-Cas9 methods followed by microarray analysis which can provide significant data on hundreds of dysregulated genes. This analysis, although very useful, lacks information about the direct control of the dysregulated genes. To further investigate these genes, In silico analysis using bioinformatics provides additional information to identify direct targets of the transcription factor studied. Additionally, the functionality of these response elements can be analyzed using site-directed mutagenesis and promoter-gene-reporter assays. We have shown recently that the oncogenic transcription factor MYC (Cellular Myelocytomatosis Oncogene) controls the expression of the ITGA1 (integrin alpha 1 subunit) gene in colorectal cancer, and that their expression correlates in 72% of colorectal tumors. This protocol describes a procedure for the analysis of the ITGA1 promoter and the identification of response elements for the MYC oncogene. The latter is known to be a member of the MYC/MAX/MAD network. Interactions between these factors lead to gene activation or repression depending on upstream signalization and cell condition.

Materials and Reagents

  1. 1.5 ml tubes
  2. 10 cm dish
  3. 12-well plates (Corning, Falcon®, catalog number: 353043 )
  4. White 96-well assay plate (Corning, catalog number: 3912 )
  5. Ice
  6. HEK293T cells (ATCC, catalog number: CRL-3216 ) at low passage
  7. DH5α competent cells
  8. Primers (Sequences of the oligonucleotide primers used for the site directed mutagenesis):
    Forward 5’-CGACTTCACGGTGAATTTGGACAATCCGCAGGGGATGGAAGG-3’
    Reverse 5’-CCTTCCATCCCCTGCGGATTGTCCAAATTCACCGTGAAGTCG-3’
  9. The promoter of interest, as for example here, the ITGA1 proximal promoter sequence inserted in the pLightSwitch_prom plasmid vector (SwitchGear Genomics, catalog number: S706788 )
  10. Plasmid constructs carrying the activator or repressor to be characterized, as for example here: pcDNA-Empty vector, pcDNA-MYC and pCMV-MAD, as described in Ni et al., 2005
  11. The pGL4.13 plasmid (luc2/SV40) as a control of transfection efficiency
  12. GeneArt Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A13282 )
  13. DNase and RNase free water
  14. Distilled water
  15. 0.5 M EDTA, pH 8
  16. SOC medium
  17. LB agar plates
  18. Trypan blue
  19. Effectene transfection reagent (QIAGEN, catalog number: 301425 )
  20. PBS
  21. DMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11995073 )
  22. Fetal bovine serum (FBS) (BOLLE COMMUNICATION, WISENT, catalog number: 080-150 )
  23. GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  24. HEPES (BOLLE COMMUNICATION, WISENT, catalog number: 330-050-EL )
  25. Dual Luciferase Reporter Assay System (Promega, catalog number: E1980 )
    Luciferase Assay substrate
    Luciferase Assay buffer
  26. DMEM culture medium for HEK293T cells (see Recipes)
  27. LAR II solution (see Recipes)
  28. 1x Stop & Glo (see Recipes)

Equipment

  1. Orion Microplate Luminometer (Berthold, Bad Wildbad, Germany)
  2. MyCycler Personal Thermal Cycler for PCR reactions (Bio-Rad Laboratories)
  3. Water bath
  4. Cell culture incubator

Software

  1. MatInspector web based software. Genomatix (Munich, Germany)
    https://www.genomatix.de/online_help/help_matinspector/matinspector_help.html
  2. Blast Global Align
    https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=blastn&BLAST_SPEC=GlobalAln&LINK_LOC=BlastHomeLink

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Boudjadi, S. and Beaulieu, J. (2017). In silico Analysis and Site-directed Mutagenesis of Promoters. Bio-protocol 7(6): e2181. DOI: 10.21769/BioProtoc.2181.

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分类

分子生物学 > DNA > 诱/突变

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