Rapid Generation of Nested Chromosomal Deletions on Mouse Chromosome 2

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-09, Vol.97 (19), p.10471-10476
Main Authors: LePage, David F., Church, Deanna M., Millie, Elise, Hassold, Terry J., Conlon, Ronald A.
Format: Article
Language:English
Subjects:
Alleles
Animals
Biological Sciences
Cell lines
chromosome 2
Chromosome Deletion
Chromosomes
Fishing lines
Genes
Genetic screening
Genetic vectors
Genetics
Germ cells
Hybrid Cells
Hybridity
Membrane Proteins - genetics
Mice
Molecular Sequence Data
Mutation
Notch1 gene
Plasmids
Receptor, Notch1
Receptors, Cell Surface
Rodents
Transcription Factors
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Summary:Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or 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 tested. 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 F1hybrid embryonic stem cell line, F1(129S1× Cast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.97.19.10471