Mutagenesis by reversible promoter insertion to study the activation of NF-κB

Eugene S. Kandel, Tao Lu, Youzhong Wan, Mukesh K. Agarwal, Mark W. Jackson, George R. Stark

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-κB-dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-κB p65 subunit, and the NF-κB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-κB-dependent secretion of factors that activate NF-κB through cell-surface receptors, establishing an autocrine loop.

Original languageEnglish (US)
Pages (from-to)6425-6430
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number18
DOIs
StatePublished - May 3 2005
Externally publishedYes

Fingerprint

Mutagenesis
Phenotype
Genes
Mutation
Insertional Mutagenesis
Cell Surface Receptors
Introns
Genetic Recombination
Dissection
Proteins
Clone Cells
Alleles
Genome
DNA

Keywords

  • Act1
  • Forward genetics
  • p65 RelA

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Mutagenesis by reversible promoter insertion to study the activation of NF-κB. / Kandel, Eugene S.; Lu, Tao; Wan, Youzhong; Agarwal, Mukesh K.; Jackson, Mark W.; Stark, George R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 18, 03.05.2005, p. 6425-6430.

Research output: Contribution to journalArticle

Kandel, Eugene S. ; Lu, Tao ; Wan, Youzhong ; Agarwal, Mukesh K. ; Jackson, Mark W. ; Stark, George R. / Mutagenesis by reversible promoter insertion to study the activation of NF-κB. In: Proceedings of the National Academy of Sciences of the United States of America. 2005 ; Vol. 102, No. 18. pp. 6425-6430.
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