Sleeping Beauty transposon system
The reconstruction of SB transposase was based on the concept that there was a primordial Tc1-like transposon that was the ancestor to the sequences found in fish genomes.
[10] The construction for the transposase began by fusing portions of two inactive transposon sequences from Atlantic salmon (Salmo salar) and one inactive transposon sequence from rainbow trout (Oncorhynchus mykiss) and then repairing small deficits in the functional domains of the transposase enzyme (Fig.
Each amino acid in the first completed transposase, called SB10, was determined by a “majority-rule consensus sequence” based on 12 partial genes found in eight fish species.
3) were to restore a complete protein by filling in gaps in the sequence and reversing termination codons that would keep the putative 360-amino acid polypeptide from being synthesized.
3) was to reverse mutations in the nuclear localization signal (NLS) that is required to import the transposase enzyme from the cytoplasm where it is made to the nucleus where it acts.
[4] SB10 transposase has been improved over the decade since its construction by increasing the consensus with a greater number of extinct Tc1 transposon sequences and testing various combinations of changes.
[18] The most recent version of SB transposase, SB100X, has about 100 times the activity of SB10 as determined by transposition assays of antibiotic-resistance genes conducted in tissue cultured human HeLa cells.
[16] The International Society for Molecular and Cell Biology and Biotechnology Protocols and Research (ISMCBBPR) named SB100X the molecule of the year for 2009 for recognition of the potential it has in future genome engineering.
[19] The transposon recognized by SB transposase was named T because it was isolated from the genome of another salmond fish, Tanichthys albonubes.
For some applications of genome engineering such as some forms of gene therapy,[26][27][28] avoiding the use of viruses is also important for social and regulatory reasons.
However, there are two major problems with most methods for delivering DNA to cellular chromosomes using plasmids, the most common form of non-viral gene delivery.
However, by using powerful promoters to regulate expression of a transgene, delivery of transposons to a few cells can provide useful levels of secreted gene products for an entire animal.
The widespread human application of gene therapy in first-world nations as well as countries with developing economies can be envisioned if the costs of the vector system are affordable.
Because the SB system is composed solely of DNA, the costs of production and delivery are considerably reduced compared to viral vectors.
The first clinical trials using SB transposons in genetically modified T cells will test the efficacy of this form of gene therapy in patients at risk of death from advanced malignancies.
Top line: A transposon, defined by the mirrored sets of red double arrows (IR/DRs) is shown as contained in another DNA molecule (e.g., a plasmid shown by the blue lines). The transposon in this example harbors an expression cassette consisting of a promoter (blue oval) that can direct transcription of the gene or other DNA sequence labeled “genetic cargo”. Middle lines: Sleeping Beauty (SB) transposase binds to the IR/DRs as shown and cuts the transposon out of the plasmid (the cut sites are indicated by the two black slashed lines in the remaining plasmid) Bottom two lines: Another DNA molecule (green) with a TA sequence can become the recipient of a transposed transposon. In the process, the TA sequence at the insertion site is duplicated.
The 360-amino acid polypeptide has three major subdomains: the amino-terminal DNA-recognition domain that is responsible for binding to the DR sequences in the mirrored IR/DR sequences of the transposon, a nuclear localization sequence (NLS), and a DDE domain that catalyzes the cut-and-paste set of reactions that comprise transposition. The DNA-recognition domain has two paired box sequences that can bind to DNA and are related to various motifs found on some transcription factors; the two paired boxes are labeled PAI and RED. The catalytic domain has the hallmark DDE (sometimes DDD) amino acids that are found in many transposase and recombinase enzymes. In addition, there is a region that is highly enriched in glycine (G) amino acids.
Step 1: Schematic of extinct Tc1/mariner -like transposons in modern salmonid genomes; x, missense mutations; S, termination mutations; F, frameshift mutations; G, major gap/missing amino acids.
Step 3: Elimination of the gap (G) and termination and frameshift mutations.
Step 4: reconstruction of the bipartite NLS sequence (orange underline).
Steps 5–8: reconstruction of the N-terminal DNA-binding domain (orange underline).
Steps 9–10: reconstruction of the catalytic domain (orange underline) including the signature DDE residues (green boxes).
