Introduction of the negative selection marker into replacement vectors by a single ligation step.
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.
Tzimagiorgis
G
G
Max-Planck-Institute for Psychiatry, Department of Neurochemistry, Martinsried, Munich, Germany.
Michaelidis
T M
TM
Lindholm
D
D
Thoenen
H
H
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D011088:DNA Ligases / Q000378:metabolism*; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005822:Genetic Vectors; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012641:Selection, Genetic; D013234:Stem Cells
C496450:Fgf5 protein, mouse; D005819:Genetic Markers; D019253:Proto-Oncogene Proteins c-bcl-2; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors; D011088:DNA Ligases