Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells

• DOI: 10.1007/bf01977348
• Publication Date: 2005-08-02T04:52:54Z
• Authors: James W. McCarrick; Jane R. Parnes; Rho H. Seong; Davor Solter; Barbara B. Knowles
• Keywords: 0301 basic medicine; 570; Stem-Cells; Antigens-CD4: ge; Stem Cells; Embryo: cy; Genetic-Vectors; Genetic Vectors; 610; DNA; Cell-Line; Embryo, Mammalian; Peptide Fragments; Cell Line; Peptide-Fragments: ge; Mice; 03 medical and health sciences; Diphtheria-Toxin: ge; Mutagenesis; SUPPORT-U-S-GOVT-P-H-S; CD4 Antigens; Animals; Diphtheria Toxin

The diphtheria toxin A-chain gene was used in a positive-negative selection gene targeting vector to alter the CD4 gene which is transcriptionally silent in mouse embryonic stem cells. Expression of the toxin gene was driven by a constitutively active enhancer, yet the targeting construct exhibited only minimal transient toxicity while enriching for targeted clones 9- to 29-fold. Germline transmission of the stem cell-derived genome was obtained. These data suggest the usefulness of this diphtheria toxin A-chain cassette in replacement-type positive-negative selection vectors. Its potential for novel applications, particularly in the enrichment for 'hit-and-run' insertion-type vectors, is discussed.