Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells

Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually an...

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Bibliographic Details
Published in:Nucleic acids research 2012-07, Vol.40 (13), p.e97-e97
Main Authors: Mayasari, N Ika, Mukougawa, Keiko, Shigeoka, Toshiaki, Kawakami, Koichi, Kawaichi, Masashi, Ishida, Yasumasa
Format: Article
Language:English
Subjects:
Animals
Cell Line
DNA Transposable Elements
Embryonic Stem Cells - metabolism
Gene Expression
Gene Knockout Techniques
Genetic Vectors
Methods Online
Mice
Mutagenesis, Insertional - methods
Poly A
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Summary:Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually and conditionally knockout the remaining 'difficult-to-trap' genes. Here, we show that transcriptionally silent genes in ES cells are severely underrepresented among the randomly trapped genes in KOMP. Our conditional poly(A)-trapping vector with a common retroviral backbone also has a strong bias to be integrated into constitutively transcribed genome loci. Most importantly, conditional gene disruption could not be successfully accomplished by using the retrovirus vector because of the frequent development of intra-vector deletions/rearrangements. We found that one of the cut and paste-type DNA transposons, Tol2, can serve as an ideal platform for gene-trap vectors that ensures identification and conditional disruption of a broad spectrum of genes in ES cells. We also solved a long-standing problem associated with multiple vector integration into the genome of a single cell by incorporating a mixture of differentially tagged Tol2 transposons. We believe our strategy indicates a straightforward approach to mass-production of conditionally disrupted alleles for genes in the target cells.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gks262