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Nucleic Acids Research, Vol 24, Issue 17 3476-3477, Copyright © 1996 by Oxford University Press

ARTICLES

Introduction of the negative selection marker into replacement vectors by a single ligation step

G Tzimagiorgis, TM Michaelidis, D Lindholm and H Thoenen
Max-Planck-Institute for Psychiatry, Department of Neurochemistry, Martinsried, Munich, Germany.

Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding laborious cloning procedures. Our results demonstrate that this fast and simple method consistently provides a high level of enrichment of appropriately targeted clones.
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