Open AccessDeletion and Replacement of the Mouse Adult β-Globin Genes by a “Plug and Socket” Repeated Targeting Strategy
Author(s) -
Peter J. Detloff,
Jada Lewis,
Simon W. M. John,
W R Shehee,
Robert Langenbach,
Nobuyo Maeda,
Oliver Smithies
Publication year - 1994
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.14.10.6936-6943.1994
Subject(s) - biology , minigene , locus (genetics) , genetics , gene targeting , gene , plasmid , microbiology and biotechnology , homologous recombination , hypoxanthine guanine phosphoribosyltransferase , mutant , rna , rna splicing
We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.