A simple Cre-loxP method for chromosomal N-terminal tagging of essential and inessential S. pombe genes

Werler, P., Hartsuiker, E. and Carr, A. M. (2003) A simple Cre-loxP method for chromosomal N-terminal tagging of essential and inessential S. pombe genes. Gene, 304. pp. 133-141. ISSN 0378-1119

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Abstract

To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates. Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest. This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site. In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype. Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase. This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter. We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.

Item Type: Article
Additional Information: GDSC42
Schools and Departments: School of Life Sciences
Depositing User: Gee Wheatley
Date Deposited: 20 Mar 2007
Last Modified: 30 Nov 2012 16:51
URI: http://sro.sussex.ac.uk/id/eprint/997
Google Scholar:25 Citations
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