Endogenous Transposases Affect Differently Sleeping Beauty and Frog Prince Transposons in Fish Cells

Abstract

Fish cells stably expressing exogenous genes have potential applications in the production of fish recombinant proteins, gene-function studies, gene-trapping, and the production of transgenic fish. However, expression of a gene of interest after random integration may be difficult to predict or control. In the past decade, major contributions have been made in vertebrate-gene transfer, by using tools derived from DNA transposons. Among them, the Sleeping Beauty (SB) and Frog Prince (FP) transposons, derived, respectively, from fish and frog genomes, mediate transposition in a large variety of cells, although with different efficiency. This study was aimed at assessing the activities of the SB and the FP transposases in fish cell lines from genetically distant species (CHSE-214, RTG-2, BF-2, EPC, and SAF-1). Their transpositional ability was evaluated by the plasmid-based excision assay, the colony formation assay, and the footprint patterns. The results reveal that while both transposases are active in all cell lines, the transposition rates and the precision of the transposition are overall higher with FP than SB. Our results also indicated a key role of cell-specific host factors in transposition, which was associated with the presence of Tc1-like endogenous transposases; this effect was more accentuated in the two salmonid cell lines transfected with SB. This result agrees with previous studies supporting the use of transposons in heterologous organisms to prevent from genomic instability and from impeding the precise activity of the exogenous transposase.

Tc1-like TransposaseThm3of Silver Carp (Hypophthalmichthys molitrix) Can Mediate Gene Transposition in the Genome of Blunt Snout Bream (Megalobrama amblycephala)

“…The Tc1 -like transposons are powerful molecular tools for transgenesis in vertebrate cells ( Gallardo-Galvez et al 2011 ; Garrels et al 2014 ). Most Tc1 -like transposons identified in fish are considered defective because they contain insertions/deletions that result in either sequence frameshifts or point mutations, which lead to a premature stop codon or a nonsense codon in the transcribed mRNA ( Radice et al 1994 ).…”