Transactivation and transrepression studies with p53.
The methods outlined in this chapter are designed to facilitate the study of the transactivation and transrepression properties of p53 (as well as p63 and p73). Once a gene of interest is identified, its presumptive promoter region can be cloned upstream of a luciferase gene in a plasmid. The most common reason for transfection experiments is to study gene expression patterns in the presence or absence of a particular gene product (e.g., p53). Three methods of transfection are outlined in this chapter: (i) cationic lipofection; (ii) calcium phosphate precipitation; and (iii) BES precipitation. The first method is ideal for the study of transactivation and transrepression properties of p53 (or other transcription factors). The last two are more suited for experiments where larger numbers of transfected cells are needed. Several examples of transfections and their respective results are provided.
Scian
Mariano J
MJ
Department of Biochemistry, Virginia Commonwealth University, Richmond, USA.
Frum
Rebecca
R
Deb
Swati Palit
SP
Deb
Sumitra
S
D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53