PCR analysis of insertion site specificity, transcription, and structural uniformity of the Lepidopteran transposable element IFP2 in the TN-368 cell genome

Abstract

The IFP2 element is a unique Lepidopteran transposon that has been associated with spontaneous Baculovirus mutants isolated following passage of the virus in the TN-368 cell line. Independent genomic representatives of IFP2 from TN-368 cells show little sequence divergence, suggesting that IFP2 was recently introduced into this genome and is highly stable. IFP2 is inserted within AT-rich regions of the TN-368 genome and targets TTAA sites. The specificity for TTAA target sites during transposition is not limited to the movement of IFP2 during an active Baculovirus infection, but is a property of its movement in uninfected cells as well. The exact origin of IFP2 remains obscure since it is found in two independently established Trichoplusia ni cell lines but not in three others, and we have not yet identified any IFP2 sequences in either field collected larvae or laboratory colonies.

A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture

“…S3 and Table S4). The preference for this Buster target feature is very similar to the TA (98.4%) and TTAA (97.6%) target site requirements that we have found for the Sleeping Beauty and piggyBac elements, respectively (44,45). The Buster target site duplication TAs are in the center of the 8-bp target site duplication (nnnTAnnn), whereas the Mariner/Tc1 TA (Sleeping Beauty) and piggyBac TTAA comprise the entire target site duplication.…”

“…A hyperactive 100× Sleeping Beauty mutant (55, 56) has been successfully isolated by site-directed mutagenesis, and is hyperactive in HeLa cells as well as murine embryonic and adult stem cells. Such hyperactive transposases facilitate transposon use as vectors in human gene therapy, regenerative medicine, and rodent genetics (44,55). As a consequence, the use of these hAT transposons should accelerate the ability to find genes involved in disease in rodent models and therefore, identify the relevant human orthologs.…”

The lepidopteran transposon vector, piggyBac , mediates germ-line transformation in the Mediterranean fruit fly

“…The element is dispersed and repeated in the genome of this cell line, but it has not been found in any others (16,20). Its presence in vivo is discontinuous among T. ni strains, and thus far, it has not been detected in any other lepidopteran or other species.…”

“…The piggyBac element is a short inverted terminal repeat (ITR) transposable element, 2.5 kb long, with 13-bp ITR sequences and a 2.1-kb ORF (15,16). It is part of a subclass of ITR elements that are thus far found only in lepidopterans and that insert exclusively into TTAA target sites (17)(18)(19).…”

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

“…It is also capable of precise excision and transposition in plasmid-based assays (Elick et al 1996a;Lobo et al 1999Lobo et al , 2001Thibault et al 1999). Assays for excision and interplasmid transposition con®rmed that piggyBac transposes via a strict``cut-and-paste'' mechanism (Elick et al 1996b;Lobo et al 1999;Thibault et al 1999), inserting exclusively at 5¢-TTAA-3¢ target sites that are duplicated upon insertion (Cary et al 1989;Wang and Fraser 1993;Fraser et al 1995;Elick et al 1996b), and excising precisely (Elick et al 1996b;Fraser et al 1996) leaving no footprint. Excision assays using both wild-type and mutagenized piggyBac terminal sequences demonstrated that the element does not discriminate between proximal and distal duplicated ends, and suggest that the transposase does not ®rst recognize an internal binding site and then scan towards the ends (Elick et al 1997).…”