Sleeping Beauty transposon system
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The Sleeping Beauty transposon system is a synthetic DNA
Mechanism of action
The Sleeping Beauty transposon system is composed of a Sleeping Beauty (SB)
As do all other Tc1/mariner-type transposases, SB transposase inserts a transposon into a TA dinucleotide base pair in a recipient DNA sequence.[2] The insertion site can be elsewhere in the same DNA molecule, or in another DNA molecule (or chromosome). In mammalian genomes, including humans, there are approximately 200 million TA sites. The TA insertion site is duplicated in the process of transposon integration. This duplication of the TA sequence is a hallmark of transposition and used to ascertain the mechanism in some experiments. However, a recent study indicated that SB also integrates into non-TA dinucleotides at a low frequency.[3] The transposase can be encoded either within the transposon (e.g., the putative transposon shown in Fig. 2) or the transposase can be supplied by another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons (e.g., Fig. 1) are most useful as genetic tools because after insertion they cannot independently continue to excise and re-insert. All of the DNA transposons identified in the human genome and other mammalian genomes are non-autonomous because even though they contain transposase genes, the genes are non-functional and unable to generate a transposase that can mobilize the transposon.
Construction
This resurrected transposase gene was named "Sleeping Beauty (SB)" because it was brought back to activity from a long evolutionary sleep.[4] The SB transposon system is synthetic in that the SB transposase was re-constructed from extinct (fossil) transposase sequences belonging to the Tc1/mariner class of transposons[5][6] found in the genomes of salmonid fish.[7] As in humans, where about 20,000 inactivated Tc1/mariner-type transposons comprise almost 3% of the human genome,[8][9] the transposase genes found in fish have been inactive for more than 10 million years due to accumulated mutations. 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. Although there were many sequences that looked like Tc-1 transposons in all the fish genomes studied, the transposon sequences were all inactive due to mutations. By assuming that the variations in sequences were due to independent mutations that accumulated in the different transposons, a putative ancestral transposon (Fig. 2) was postulated.[10]
The construction for the transposase began by fusing portions of two inactive transposon sequences from
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.[12][13][14][15][16][17] Further work has shown that the DNA-binding domain consists of two paired sequences, which are homologous to sequence motifs found in certain transcription factors. The paired subdomains in SB transposase were designated PAI and RED. The PAI subdomain plays a dominant role in recognition of the DR sequences in the transposon. The RED subdomain overlaps with the nuclear localization signal, but its function remains unclear.[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. The transposon consists of a genetic sequence of interest that is flanked by
Applications
Over the past decade, SB transposons have been developed as non-viral vectors for introduction of genes into genomes of vertebrate animals and for
Figure 4 illustrates these two uses of SB transposons.For either gene delivery or gene disruption, SB transposons combine the advantages of viruses and naked DNA. Viruses have been evolutionarily selected based on their abilities to infect and replicate in new host cells. Simultaneously, cells have evolved major molecular defense mechanisms to protect themselves against viral infections. 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. The use of non-viral vectors avoids many, but not all, of the defenses that cells employ against vectors.
Arguably the most exciting potential application of Sleeping Beauty transposons will be for human gene therapy. 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.[31]
See also
References
- S2CID 28784300.
- PMID 10431195.
- PMID 29445422.
- ^ PMID 9390559.
- PMID 8302872.
- S2CID 11381762.
- PMID 8051704.
- PMID 11181995.
- PMID 11237011.
- PMID 8643520.
- S2CID 29930180.
- PMID 12842434.
- PMID 14759813.
- PMID 15456893.
- PMID 16150650.
- ^ S2CID 27373372.
- PMID 20372108.
- PMID 12082109.
- ^ Vence T. ""Sleeping Beauty" named Molecule of the Year". mdc-berlin.de. Retrieved 10 May 2011.
- ^ PMID 12083513.
- PMID 10964563.
- S2CID 3194633.
- PMID 20478384.
- PMID 15680506.
- PMID 20869786.
- PMID 14759798.
- PMID 16096013.
- PMID 21459777.
- PMID 17407189.
- PMID 19384290.
- PMID 20104209.