Nucleic Acids Research, 1991, Vol. 19, No. 10 2637-2643
© 1991
Modulation of gene activity by consecutive gene targeting of one creatine kinase M allele in mouse embryonic stem cells
Jan van Deursen,
Robin Lovell-Badge1,
Frank Oerlemans,
Jan Schepens and
Bé Wieringa*
Department of Cell Biology and Histology, University Nijmegen PO Box 9101, 6500HB Nijmegen, The Netherlands
1Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research Mill Hill, London NW7 1AA, UK
* To whom correspondence should be addressed
Received February 26, 1991. Revised April 15, 1991. Accepted April 15, 1991.
The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptlonally Inactive during early embryogenesis. Using repeated rounds of gene targeting In mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an Insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several Independent doubly targeted clones were isolated which all had insertions in the previously neo-dlsrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted In gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels In ES cells, led to the formation of the BM isoform of CK in these clones.
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