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.
Highlights
The natural medaka fish hAT gene family element Tol2 [1,2,3], the engineered Tc1/mariner transposons Sleeping Beauty (SB) [4] and Frog Prince [5], and the insect-derived natural element PiggyBac [6] represent transposons potentially suitable as DNA transfer tools for gene discovery and gene delivery applications in vertebrates
While testing green fluorescent protein (GFP)-marked Tol2 vectors in zebrafish, we noticed an unusually high number of GFP-positive cells in embryos injected with Tol2 transposon and synthetic transposase mRNA derived from an updated Tol2 transcription vector (Figure 1), suggesting that this transposon is highly active in somatic tissues
We found that increased GFP fluorescence in Tol2-injected embryos versus SB-injected embryos correlated with transposon excision
Summary
The natural medaka fish hAT gene family element Tol2 [1,2,3], the engineered Tc1/mariner transposons Sleeping Beauty (SB) [4] and Frog Prince [5], and the insect-derived natural element PiggyBac [6] represent transposons potentially suitable as DNA transfer tools for gene discovery and gene delivery applications in vertebrates. We have been studying the Sleeping Beauty transposable element for molecular genetic applications in vertebrates. The availability of an alternative, active transposon system devoid of these disadvantages and adapted for use in higher vertebrates would offer tremendous potential uses in a variety of molecular genetic and biotechnological fields. There is a potential for synergism between multiple transposon systems, both in gene discovery and in gene transfer applications. Gene transfer into cultured human cells or live mammals using Tol has not been previously reported, nor have the transposon sequence requirements for Tol2-based transposition been previously well characterized
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