Rapid generation of nested chromosomal deletions on mouse chromosome 2

Citation
Df. Lepage et al., Rapid generation of nested chromosomal deletions on mouse chromosome 2, P NAS US, 97(19), 2000, pp. 10471-10476
Citations number
38
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
0027-8424 → ACNP
Volume
97
Issue
19
Year of publication
2000
Pages
10471 - 10476
Database
ISI
SICI code
0027-8424(20000912)97:19<10471:RGONCD>2.0.ZU;2-6
Abstract
Nested chromosomal deletions are powerful genetic tools. They are particula rly suited for identifying essential genes in development either directly o r by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and test ed. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre, Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F-1 hybrid embryonic stem c ell line, F-1(129S1 xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extend ing six or seven centiMorgans. The cytology of the deletion chromosomes wer e determined by fluorescent in site hybridization, Eight deletions were cyt ologically normal, but the two largest deletions had additional rearrangeme nts. Three deletions, including the largest unrearranged deletion, have bee n transmitted through the germ line. Several endpoints also have been clone d by plasmid rescue. These experiments illustrate the means to rapidly crea te and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.