Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.

TLDR

The authors engineered promoter‑trap constructs lacking a promoter but containing a beta‑galactosidase reporter, such that insertion into an active transcription unit both disrupts the target gene and activates reporter expression, and introduced these constructs into embryonic stem cells by electroporation or retroviral infection to generate transgenic lines. Transgenic lines derived from these promoter‑trap constructs displayed diverse beta‑galactosidase expression patterns, and intercrossing heterozygotes from 24 strains revealed that homozygosity in 9 strains caused embryonic lethality while the remaining strains showed no overt phenotype, indicating that many genes identified by this approach are non‑essential for development.

Abstract

A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.