Sleeping Beauty transposon donor pT4 with CAST-5/wt hybrid motifs in the inner 12DRs
Sleeping Beauty transposon donor whose two inner 12DR transposase-binding sites carry a CASTing-selected high-affinity CAST-5/wt hybrid motif in place of the wild-type PAI recognition motif. Transposes at 175% of the wild-type donor in a colony-based assay in HeLa cells.
Origin: Engineered from the Sleeping Beauty pT/neo donor
Characteristics
Built by PCR mutagenesis of pT/neo, a neomycin-marked Sleeping Beauty donor. The CAST-5/wt hybrid replaces only the PAI interaction motif of the inner 12DRs and keeps the rest of each DR wild type. CAST-5 came from six rounds of CASTing with recombinant SB transposase, and some CASTing-selected sequences were bound up to eight-fold more strongly than the wild-type 14DR by the PAIRED domain in EMSA. Transposition is 175% of the wild-type donor (n=4, G418-resistant colonies in HeLa cells).
Applications: Donor for Sleeping Beauty transposition into the genome of cultured human HeLa cells, scored as G418-resistant colonies after cotransfection with a transposase plasmid, where it outperforms the wild-type donor 1.75-fold. Reported as a way to raise the efficiency of Sleeping Beauty transposition for genetic applications.
Limitations: The same CAST-5/wt motif at the outer 14DRs or at inner and outer DRs lowers transposition, and full CAST-5 motifs at the inner 12DRs give 65% of wild type. Transposition by Sleeping Beauty declines as transposon size grows. The cited paper uses SB100X only in GFP excision assays of shortened transposons, and pT4 with SB100X is not reported.
References
- Wang et al. (2017). Regulated complex assembly safeguards the fidelity of Sleeping Beauty transposition. Nucleic Acids Res - Wang 2017 pT4 Transposon