Harnessing a High Cargo-Capacity Transposon for Genetic Applications in Vertebrates

Abstract

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.

The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes

“…The piggyBac element has been shown to serve as a gene-transfer vector when the entire size is 14.3 kb (Ding et al 2005). In the case of the Tol2 element, the maximum element size so far reported is 10.2 kb (Balciunas et al 2006). It is possible that the latter two elements retain transposition activity when they are longer than the reported sizes.…”

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis‐regulatory regions in zebrafish

“…This vector was generated by first cloning a fragment containing the Cardiac Actin promoter driving GFP from the pCARGFP vector (Kroll and Amaya, 1996) into the XbaI/NotI restriction sites of the pminiTol2/MCS vector (Balciunas et al, 2006) to generate the pminiTol2-CARGFP. The Z48 enhancer was then PCR amplified from zebrafish DNA using the primers 5Ј-GGTCTAGAGCTCTCGCAGTTGT-GGGC-3Ј and 5Ј-CCTCTAGAGGTAC CCCCCCTGCTTAAGACACAG-3Ј, which contain a XbaI restriction sites in the forward primer and a XbaI and KpnI restriction sites in the reverse one (underlined).…”

Efficient Transposition of Tol2 in the Mouse Germline

“…Unlike other DNA-type transposons like Sleeping Beauty (SB) (Ivics et al 1997) or piggyBac (PB) (Fraser et al 1996), Tol2 does not exhibit any known strong site specificity for integration nor does it exhibit any significant overexpression inhibition activity (Kawakami and Noda 2004;Balciunas et al 2006) as seen in SB (Geurts et al 2003). Recently, Tol2 was shown to effectively carry large DNA cargo of up to 10 kb in human and mouse cells without affecting its transposition efficiency (Balciunas et al 2006). To date, Tol2 has also been demonstrated to transpose efficiently in zebrafish, frog, chicken, mouse cells, and human cells (Kawakami et al 2000Koga et al 2003;Kawakami and Noda 2004;Balciunas et al 2006;Hamlet et al 2006;Sato et al 2007).…”