Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-10
2002-12-03
Crouch, Deborah (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023500, C530S350000, C435S325000, C435S440000, C435S445000
Reexamination Certificate
active
06489458
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for gene expression, mapping genes, mutagenesis, methods for introducing DNA into a host chromosome and to transposons and transposases.
Transposons or transposable elements include a short piece of nucleic acid bounded by repeat sequences. Active transposons encode enzymes that facilitate the insertion of the nucleic acid into DNA sequences.
In vertebrates, the discovery of DNA-transposons, mobile elements that move via a DNA intermediate, is relatively recent (Radice, A. D., et al., 1994.
Mol. Gen. Genet.
244, 606-612). Since then, inactive, highly mutated members of the Tc1/mariner as well as the hAT (hobo/Ac/Tam) superfamilies of eukaryotic transposons have been isolated from different fish species, Xenopus and human genomes (Oosumi et al., 1995.
Nature
378, 873; Ivics et al. 1995.
Mol. Gen. Genet.
247, 312-322; Koga et al., 1996.
Nature
383, 30; Lam et al., 1996.
J. Mol. Biol.
257, 359-366 and Lam, W. L., et al.
Proc. Natl. Acad Sci. USA
93, 10870-10875).
These transposable elements transpose through a cut-and-paste mechanism; the element-encoded transposase catalyzes the excision of the transposon from its original location and promotes its reintegration elsewhere in the genome (Plasterk, 1996
Curr. Top. Microbiol. Immunol.
204, 125-143). Autonomous members of a transposon family can express an active transposase, the trans-acting factor for transposition, and thus are capable of transposing on their own. Nonautonomous elements have mutated transposase genes but may retain cis-acting DNA sequences. These cis-acting DNA sequences are also referred to as inverted terminal repeats. Some inverted repeat sequences include one or more direct repeat sequences. These sequences usually are embedded in the terminal inverted repeats (IRs) of the elements, which are required for mobilization in the presence of a complementary transposase from another element.
Not a single autonomous element has been isolated from vertebrates; all transposon-like sequences are defective, apparently as a result of a process called “vertical inactivation” (Lohe et al., 1995
Mol. Biol. Evol.
12, 62-72). According to one phylogenetic model (Hartl et al., 1997
Trends Genet.
13, 197-201), the ratio of nonautonomous to autonomous elements in eukaryotic genomes increases as a result of the trans-complementary nature of transposition. This process leads to a state where the ultimate disappearance of active, transposase-producing copies in a genome is inevitable. Consequently, DNA-transposons can be viewed as transitory components of genomes which, in order to avoid extinction, must find ways to establish themselves in a new host. Indeed, horizontal gene transmission between species is thought to be one of the important processes in the evolution of transposons (Lohe et al., 1995 supra and Kidwell, 1992.
Curr. Opin. Genet Dev.
2, 868-873).
The natural process of horizontal gene transfer can be mimicked under laboratory conditions. In plants, transposable elements of the Ac/Ds and Spm families have been routinely introduced into heterologous species (Osborne and Baker, 1995
Curr. Opin. Cell Biol.
7, 406-413). In animals, however, a major obstacle to the transfer of an active transposon system from one species to another has been that of species-specificity of transposition due to the requirement for factors produced by the natural host. For this reason, attempts have been unsuccessful to use the P element transposon of
Drosophila melanogaster
for genetic transformation of non-drosophilid insects, zebrafish and mammalian cells (Gibbs et al., 1994
Mol. Mar. Biol. Biotech.
3, 317-326; Handler et al., 1993.
Arch. Insect Biochem. Physiol.
22, 373-384; and Rio et al., 1988
J. Mol. Biol.
200, 411-415). In contrast to P elements, members of the Tc1/mariner superfamily of transposable elements may not be as demanding for species-specific factors for their transposition. These elements are widespread in nature, ranging from single-cellular organisms to humans (Plasterk, 1996, supra). In addition, recombinant Tc1 and mariner transposases expressed in
E. coli
are sufficient to catalyze transposition in vitro (Vos et al, 1996
Genes. Dev.
10, 755-761 and Lampe et al., 1996.
EMBO J.
15, 5470-5479 and PCT International Publication No. WO 97/29202 to Plasterk et al.). Furthermore, gene vectors based on Minos, a Tc1-like element (TcE) endogenous to
Drosophila hydei
, were successfully used for germline transformation of the fly
Ceratitis capitata
(Loukeris et al., 1995
Science
270, 2002-2005).
Molecular phylogenetic analyses have shown that the majority of the fish TcEs can be classified into three major types: zebrafish-, salmonid- and Xenopus TXr-type elements, of which the salmonid subfamily is probably the youngest and thus most recently active (Ivics et al., 1996,
Proc. Natl. Acad. Sci. USA
93, 5008-5013). In addition, examination of the phylogeny of salmonid TcEs and that of their host species provides important clues about the ability of this particular subfamily of elements to invade and establish permanent residences in naive genomes through horizontal transfer, even over relatively large evolutionary distances.
TcEs from teleost fish (Goodier and Davidson, 1994
J. Mol. Biol.
241, 26-34 and Izsvak et al., 1995.
Mol. Gen. Genet.
247, 312-322), including Tdr1 in zebrafish (Izsvak et al., 1995, supra) and other closely related TcEs from nine additional fish species (Ivics et al., 1996.
Proc. Natl. Acad. Sci. USA
93, 5008-5013) are by far the best characterized of all the DNA-transposons known in vertebrates. Fish elements, and other TcEs in general, are typified by a single gene encoding a transposase enzyme flanked by inverted repeat sequences. Unfortunately, all the fish elements isolated so far are inactive due to one or more mutations in the transposase genes.
Methods for introducing DNA into a cell are known. These include, but are not limited to, DNA condensing reagents such as calcium phosphate, polyethylene glycol, and the like), lipid-containing reagents, such as liposomes, multi-lamellar vesicles, and the like, and virus-mediated strategies. These methods all have their limitations. For example, there are size constraints associated with DNA condensing reagents and virus-mediated strategies. Further, the amount of nucleic acid that can be introduced into a cell is limited in virus strategies. Not all methods facilitate integration of the delivered nucleic acid into cellular nucleic acid and while DNA condensing methods and lipid-containing reagents are relatively easy to prepare, the incorporation of nucleic acid into viral vectors can be labor intensive. Moreover, virus-mediated strategies can be cell-type or tissue-type specific and the use of virus-mediated strategies can create immunologic problems when used in vivo.
There remains a need for new methods for introducing DNA into a cell, particularly methods that promote the efficient integration of nucleic acid fragments of varying sizes into the nucleic acid of a cell, particularly the integration of DNA into the genome of a cell.
SUMMARY OF THE INVENTION
We have developed a DNA-based transposon system for genome manipulation in vertebrates. Members of the Tc1/mariner superfamily of transposons are prevalent components of the genomes of teleost fish as well as a variety of other vertebrates. However, all the elements isolated from nature appear to be transpositionally inactive. Molecular phylogenetic data were used to identify a family of synthetic, salmonid-type Tc1-like transposases (SB) with their recognition sites that facilitate transposition. A consensus sequence of a putative transposase gene was first derived from inactive elements of the salmonid subfamily of elements from eight species of fish and then engineered by eliminating the mutations that rendered these elements inactive. A transposase was created in which functional domains were identified and tested for biochemical functions individually as well as in the context of a full-length tran
Hackett Perry B.
Ivics Zoltan
Izsvak Zsuzsanna
Crouch Deborah
Mueting Raasch & Gebhardt, P.A.
Regents of the University of Minnesota
Woitach Joseph
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