A lacZ-hygromycin fusion gene and its use in a gene trap vector for marking embryonic stem cells

• Published in: Nucleic acids research Vol. 23; no. 19; pp. 4003 - 4004
• Main Authors: Natarajan, Dipa, Boulter, Catherine A.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 11.10.1995
• Subjects: Animals; beta-Galactosidase - genetics; Cell Line; Cinnamates; Embryo, Mammalian; Genetic Markers; Genetic Vectors; Hygromycin B - analogs & derivatives; Mammalia; Mice; Recombinant Fusion Proteins; Restriction Mapping; Stem Cells
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/23.19.4003

The use of gene trap vectors in embryonic stem (ES) cells and transgenic mice has proved to be a powerful method for identifying new genes involved in mammalian development. Generally, they have a lacZ marker gene downstream of a splice acceptor site and rely on integration into introns for their expression. Not only can insertion of these vectors cause disruption of the gene into which they integrate, but it is also possible to visualize the transcription pattern of the disrupted gene itself, by staining with X-gal for expression of beta -galactosidase from the lacZ gene. More sophisticated gene trap vectors have now been developed carrying a lacZ-neomycin fusion gene, beta geo, which produces a chimaeric fusion protein with both beta -galactosidase and neomycin phosphotransferase activities, conferring the added advantage of selection with G418 for insertion within expressed genes. Such vectors can also be used for cell marking experiments by integrating within constitutively active genes. Recently beta geo gene traps have been successfully used to mark ES cells for in situ localization of their derivatives in chimaeric mice, providing a useful tool for mosaic analysis in vivo. The usefulness of beta geo gene trap vectors for cell marking is however limited to cell lines that do not already carry a neomycin phosphotransferase (neo) gene and are thus not resistant to G418. As neo is widely used as a selectable marker for mammalian cells, this excludes the majority of cells carrying expression constructs, such as ES cell lines that have undergone gene targeting events. We have therefore created a novel gene fusion between the lacZ and hygromycin B phosphotransferase (hyg) genes, named beta gyg, and have used it to construct a gene trap vector which carries this gene downstream of the splice acceptor site. Here we show that the fusion protein produced by this vector has both beta -galactosidase and hygromycin phosphotransferase activities. Moreover, we have successfully introduced this vector into a G418 resistant ES cell line, SR2-3, and by selecting for hygromycin expression, have isolated marked clones that constitutively express beta -galactosidase in all cells both before and after differentiation in vitro.