User:Perryhackett

Perry Hackett has been active in molecular genetics since 1963. His undergraduate degree is in Physics from Stanford University and he has a Ph.D., in Biophysics and Genetics from the University of Colorado (interrupted by military service). He postdoced at the Max Planck Institute for Cell Biology in Germany and at the University of California in San Francisco with Mike Bishop and Harold Varmus. Since 1980 he has been a professor at the University of Minnesota in the Department of Genetics, Cell Biology and Development and is member of the Center for Genome Engineering, the Institute of Human Genetics, and the Masonic Cancer Center. His research has focused on molecular genetics, functional genomics and transgenic organisms, with a concentration on retroviruses and transposons as vectors for delivering genes into the genomes of recipient organisms. He has used bacteria, fish, mice and dogs as model systems for investigating various transgenic technologies in vertebrate animals. The Sleeping Beauty Transposon System was invented in his lab as a way of delivering genes to vertebrates and as a way of tagging genes to determine their functions. The Sleeping Beauty Transposon System is being tested as a vector for human gene therapy and other biomedical technologies as well as for engineering animals for commercial purposes.

Sleeping Beauty Transposon System

Contents

1.	Mechanism of Action

2.	Construction of the SB system

3.	Applications

4.	References

The Sleeping Beauty transposon system is a synthetic DNA transposon that was constructed to introduce precisely defined DNA sequences into the chromosomes of vertebrate animals for the purposes of introducing new traits and to discover new genes and their functions.

1. Mechanism of Action

The Sleeping Beauty transposon system is composed of a Sleeping Beauty (SB) transposase and a transposon that was designed in 1997 to insert specific sequences of DNA into genomes of vertebrate animals. DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner (Fig. 1). Transposition is a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome. *

As do all other Tc1/mariner-type transposases, SB transposase inserts a transposon into a TA dinucleotide base pair in a recipient DNA sequence. 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. 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.

2. Construction of the SB Transposon System

Drs. Zoltan Ivics and Zsuzsanna Izsvak, working in Perry Hackett’s laboratory at the University of Minnesota, named the resurrected transposase gene Sleeping Beauty, because they brought it back to activity from a long evolutionary sleep. 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 found in the genomes of salmonid fish. As in humans, where about 20,000 inactivated Tc1/mariner-type transposons comprise almost 3% of the human genome, 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 predicted.

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. 3). 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. The first steps (1->3 in Fig. 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. The next step (4 in Fig. 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. The amino-terminus of the transposase, which contains the DNA-binding motifs for recognition of the direct repeats (DRs), was restored in steps 5->8. The last two steps restored the catalytic domain, which features conserved aspartic acid (D) and glutamic acid (E) amino acids with specific spacing that are found in integrases and recombinases. The end result was SB10, which contains all of the motifs required for function.

SB10 transposase has been improved over the decade since its construction by increasing the consensus with a greater number of extinct transposon sequences and testing various combinations of changes. 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 is remains unclear. 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. 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 (http://www.biotechniques.com/news/Sleeping-Beauty-named-Molecule-of-the-Year/biotechniques-187068.html).

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 inverted repeats (IRs) that themselves contain short direct repeats (DR) (tandem arrowheads IR-DR in Figs. 1 and 2). T had the closest IR/DR sequence to the consensus sequence for the extinct Tc-1 like transposons in fish. The consensus transposon has IRs of 231 base pairs. The innermost DRs are 29 base pairs long whereas the outermost DRs are 31 base pairs long. The difference in length is critical for maximal transposition rates. The original T transposon component of the SB transposon system has been improved with minor changes to conform to the consensus of many related extinct and active transposons.

3. Applications of SB transposons

Over the past decade, SB transposons have been developed as non-viral vectors for introduction of genes into genomes of vertebrate animals and for gene therapy. The genetic cargo can be an expression cassette - a transgene and associated elements that confer transcriptional regulation for expression at a desired level in specific tissue(s). An alternative use of SB transposons is to discover functions of genes, especially those that cause cancer, by delivering DNA sequences that maximally disrupt expression of genes close to the insertion site. This process is referred to as insertional mutagenesis or transposon mutagenesis. When a gene is inactivated by insertion of a transposon (or other mechanism), that gene is “knocked out”. Knockout mice and knockout rats have been made with the SB system. 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 , avoiding the use of viruses is also important for social and/or regulatory reasons. The use of non-viral vectors avoids many, but not all, of the defenses that cells employ against vectors.

Plasmids, the circular DNAs shown in Fig. 1, are generally used for non-viral gene delivery. 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. First, expression of transgenes from plasmids is brief due to lack of integration and due to cellular responses that turn off expression. Second, uptake of plasmid molecules into cells is difficult and inefficient. The Sleeping Beauty Transposon System was engineered to overcome the first problem. DNA transposons precisely insert defined DNA sequences (Fig. 1) almost randomly into host genomes thereby increasing the longevity of gene expression (even through multiple generations). Moreover, transposition avoids the formation of multiple, tandem integrations, which often results in switching off expression of the transgene. Currently, insertion of transgenes into chromosomes using plasmids is much less efficient than using viruses. 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.

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 comprised 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.

4. REFERENCES