title: Mixture of differentially tagged Tol2 transposons accelerates conditional disruption of a broad spectrum of genes in mouse embryonic stem cells.
abstract: 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.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan.
authors.forename: N Ika
authors.initials: NI
authors.lastname: Mayasari
authors.forename: Keiko
authors.initials: K
authors.lastname: Mukougawa
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Shigeoka
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
authors.forename: Masashi
authors.initials: M
authors.lastname: Kawaichi
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Ishida
mesh_terms: D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements; D053595:Embryonic Stem Cells / Q000378:metabolism*; D015870:Gene Expression; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D011061:Poly A
chemical_list: D004251:DNA Transposable Elements; D011061:Poly A