Tol2 transposase from medaka fish

DNA transposase from medaka fish that catalyzes precise cut-and-paste transposition with large cargo capacity. Member of the hAT (hobo/Ac/Tam3) transposable element family originally identified in Oryzias latipes. Creates 8 bp target site duplications upon genomic integration. Highly efficient in zebrafish, mouse, human, and Xenopus cells without overexpression inhibition.

Length: 1950 bp(650 aa)

Recognition site: Terminal inverted repeats (TIRs)

Directionality: Unidirectional

Efficiency: >50% germline transmission in zebrafish F0 founders

Origin: Oryzias latipes (medaka fish)

Characteristics

Recognizes and binds terminal inverted repeat sequences (TIRs) flanking transposon cargo. Catalyzes precise excision from donor plasmid and genomic integration via cut-and-paste mechanism. Creates characteristic 8 bp target site duplications flanking integrated transposon. Maintains high transposition activity across wide range of transposon:transposase ratios (1:1 to 1:10), showing no overexpression inhibition unlike Sleeping Beauty. Large cargo capacity supports efficient transposition of >10 kb constructs with activity comparable to smaller 5 kb transposons. Active transposition demonstrated in zebrafish germline, mouse ES cells, human HT1080 cells, and Xenopus embryos.

Applications: Stable transgene integration in zebrafish, mouse, and other vertebrate models for functional genomics. Gene trap and enhancer trap screens for developmental genetics research. Generation of transgene-free induced pluripotent stem cells via transient transposase expression followed by transposon excision. Hydrodynamic gene delivery to mouse liver for hereditary tyrosinemia and other gene therapy applications. Tol2kit Gateway cloning system enables modular assembly of complex transgenic constructs. Germline transgenesis with high founder transmission rates exceeding 50% in zebrafish.

Limitations: Requires terminal inverted repeat sequences flanking cargo for transposition activity. Mosaic integration patterns typical in F0 injections require breeding to establish stable germline-transmitted lines. Integration site preferences may lead to local hopping rather than random genomic distribution in some contexts. Cut-and-paste mechanism excises transposon from donor plasmid, potentially leaving small footprints at excision sites in rare cases.

Sequence

atggaggaagtatgtgattcatcagcagctgcgagcagcacagtccaaaatcagccacaggatcaagagcacccgtggccgtatcttcgcgaattcttttctttaagtggtgtaaataaagattcattcaagatgaaatgtgtcctctgtctcccgcttaataaagaaatatcggccttcaaaagttcgccatcaaacctaaggaagcatattgagagaatgcacccaaattacctcaaaaactactctaaattgacagcacagaagagaaagatcgggacctccacccatgcttccagcagtaagcaactgaaagttgactcagttttcccagtcaaacatgtgtctccagtcactgtgaacaaagctatattaaggtacatcattcaaggacttcatcctttcagcactgttgatctgccatcatttaaagagctgattagtacactgcagcctggcatttctgtcattacaaggcctactttacgctccaagatagctgaagctgctctgatcatgaaacagaaagtgactgctgccatgagtgaagttgaatggattgcaaccacaacggattgttggactgcacgtagaaagtcattcattggtgtaactgctcactggatcaaccctggaagtcttgaaagacattccgctgcacttgcctgcaaaagattaatgggctctcatacttttgaggtactggccagtgccatgaatgatatccactcagagtatgaaatacgtgacaaggttgtttgcacaaccacagacagtggttccaactttatgaaggctttcagagtttttggtgtggaaaacaatgatatcgagactgaggcaagaaggtgtgaaagtgatgacactgattctgaaggctgtggtgagggaagtgatggtgtggaattccaagatgcctcacgagtcctggaccaagacgatggcttcgaattccagctaccaaaacatcaaaagtgtgcctgtcacttacttaacctagtctcaagcgttgatgcccaaaaagctctctcaaatgagcactacaagaaactctacagatctgtctttggcaaatgccaagctttatggaataaaagcagccgatcggctctagcagctgaagctgttgaatcagaaagccggcttcagcttttaaggccaaaccaaacgcggtggaattcaacttttatggctgttgacagaattcttcaaatttgcaaagaagcaggagaaggcgcacttcggaatatatgcacctctcttgaggttccaatgtttaatccagcagaaatgctgttcttgacagagtgggccaacacaatgcgtccagttgcaaaagtactcgacatcttgcaagcggaaacgaatacacagctggggtggctgctgcctagtgtccatcagttaagcttgaaacttcagcgactccaccattctctcaggtactgtgacccacttgtggatgccctacaacaaggaatccaaacacgattcaagcatatgtttgaagatcctgagatcatagcagctgccatccttctccctaaatttcggacctcttggacaaatgatgaaaccatcataaaacgaggcatggactacatcagagtgcatctggagcctttggaccacaagaaggaattggccaacagttcatctgatgatgaagattttttcgcttctttgaaaccgacaacacatgaagccagcaaagagttggatggatatctggcctgtgtttcagacaccagggagtctctgctcacgtttcctgctatttgcagcctctctatcaagactaatacacctcttcccgcatcggctgcctgtgagaggcttttcagcactgcaggattgcttttcagccccaaaagagctaggcttgacactaacaattttgagaatcagcttctactgaagttaaatctgaggttttacaactttgagtag

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References

Kawakami et al. (2000). Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci USA - Kawakami 2000 Tol2 Transposase
Kawakami et al. (1999). Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene - Kawakami 1999 Tol2 Identification
Kawakami et al. (2004). Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics - Kawakami 2004 Mouse Transposition
Kawakami et al. (2007). Tol2: a versatile gene transfer vector in vertebrates. Genome Biol - Kawakami 2007 Tol2 Review