Citation

  • Authors: Helou L. et al.
  • Year: 2021
  • Journal: J Mol Biol 433 166839
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: G401

Method

Briefly, each sample of 100,000 cells in a well of a 24-well plates of plaque assays was transfected with jetOptimus and 500 ng DNA plasmid (pBSK-IFP2-TIR5′-NeoR-TIR3′, pBSK-Tcr-pble-TIR5′-NeoR-TIR3′ or pBSK-NeoR) as recommended by the supplier (Polyplus- transfection, Illkirch-Graffenstaden). Two days post-transfection, each cell sample was transferred to a cell culture dish (100 mm diameter) and selected with a culture medium containing 2 mg/mL G418 sulfate (Eurobio Scientific, Les Ulis) for 15 days. After two washing with 1X saline phosphate buffer, cell clones were fixed and stained overnight with 70% EtOH-0.5% methylene blue and colonies > 0.5 mm in diameter were counted. Experiments were performed at least twice in triplicate.

Abstract

The vertebrate piggyBac derived transposase 5 (PGBD5) encodes a domesticated transposase, which is active and able to transpose its distantly related piggyBac-like element (pble), Ifp2. This raised the question whether PGBD5 would be more effective at mobilizing a phylogenetically closely related pble element. We aimed to identify the pble most closely related to the pgbd5 gene. We updated the landscape of vertebrate pgbd genes to develop efficient filters and identify the most closely related pble to each of these genes. We found that Tcr-pble is phylogenetically the closest pble to the pgbd5 gene. Furthermore, we evaluated the capacity of two murine and human PGBD5 isoforms, Mm523 and Hs524, to transpose both Tcr-pble and Ifp2 elements. We found that both pbles could be transposed by Mm523 with similar efficiency. However, integrations of both pbles occurred through both proper transposition and improper PGBD5-dependent recombination. This suggested that the ability of PGBD5 to bind both pbles may not be based on the primary sequence of element ends, but may involve recognition of inner DNA motifs, possibly related to palindromic repeats. In agreement with this hypothesis, we identified internal palindromic repeats near the end of 24 pble sequences, which display distinct sequences.

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