Improved "activator trap" method for the isolation of transcriptional activation domains from random DNA fragments.
We have previously developed an "activator trap" method for selective isolation of activation domains from viral and cellular transcription factors. In this method, random sonicated DNA fragments were ligated next to the DNA binding domain (DBD) of the GAL4 factor in a plasmid that also contained a simian virus 40 (SV40) replication origin. A library of such random insert plasmids was transfected into a monkey cell line (CV-1-5GT), which had been stably transformed with a GAL4-inducible SV40 T-antigen gene. Chimeric GAL factors with a heterologous activation domain were harvested after selective replication in these CV-1-5GT cells. Here we report a simplification and generalization of the "activator trap" method. First, the time-consuming library construction step can be omitted by direct transfection of the sonicated DNA fragments and the linearized recipient plasmid vector into CV-1-5GT cells to obtain chimeric GAL-activation factors by in vivo ligations. Second, the dependence on CV-1-5GT cells can be bypassed by direct co-transfection of all components, including a plasmid carrying the T-antigen gene into cells other than CV-1-5GT. This latter step allows the application of the method to cultured human cells, as demonstrated with the human B-cell line BJA-B.
Escher
D
D
Universität Zürich, Switzerland. escher@molbio2.unizh.ch
Schaffner
W
W
D000818:Animals; D002460:Cell Line; D006801:Humans; D015533:Transcriptional Activation; D014162:Transfection