US20140038292A1

US20140038292A1 - Tol1 FACTOR TRANSPOSASE AND DNA INTRODUCTION SYSTEM USING THE SAME

Info

Publication number: US20140038292A1
Application number: US14/047,550
Authority: US
Inventors: Akihiko Koga; Satoshi Hamaguchi
Original assignee: Nagoya University NUC; Niigata University NUC
Current assignee: Nagoya University NUC; Niigata University NUC
Priority date: 2006-12-13
Filing date: 2013-10-07
Publication date: 2014-02-06
Also published as: JPWO2008072540A1; JP5320546B2; US8598328B2; WO2008072540A1; US20100129914A1

Abstract

An object is to provide a Tol1 element transposase and a use thereof. Provided is a Tol1 element transposase containing (a) a protein having the amino acid sequence of SEQ ID No: 1 or (b) a protein having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 1 and having an enzymatic activity for transferring Tol1 element. Further, provided is a polynucleotide encoding the transposase and an expression construct containing the polynucleotide therein. The present invention also provides a DNA introduction system including (a) a donor factor having such a structure that a desired DNA is inserted in a transposase gene-defected Tol1 element and (b) a helper factor containing the transposase or the polynucleotide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Ser. No. 12/518,967, filed Jan. 5, 2010 and published as U.S. 2010-0129914 A1 on May 27, 2010. U.S. Ser. No. 12/518,967 is the U.S. national phase, pursuant to 35 U.S.C. §371, of PCT international application Ser. No. PCT/JP2007/073565, filed Dec. 6, 2007, designating the United States and published in Japanese on Jun. 19, 2008 as publication WO 2008/072540. PCT/JP2007/073565 claims priority to Japanese Patent Application Ser. No. 2007-253321, filed Sep. 28, 2007 and to Japanese Patent Application Ser. No. 2006-335786, filed Dec. 13, 2006. The entire contents of the aforementioned patent applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 7, 2013, is named 84239DIV305882_ST25.txt and is 106,496 bytes in size.

TECHNICAL FIELD

The present invention relates to an enzyme for catalyzing transposition of a transposon (hereinafter referred to as a transposase) and uses thereof. Specifically, the present invention relates to a transposase of Tol1 element (Transposable element of Oryzias latipes, no. 1) which is a transposon derived from medaka fish. The invention also relates to a polynucleotide encoding the transposase, a DNA introduction system and a DNA introduction method using the transposase, and a DNA introducing kit using the system.

BACKGROUND ART

A DNA transposable element is one kind of DNA repeat sequence, and are common invertebrate genomes. However, most vertebrate transposable elements have lost their transposition activities. Among vertebrate DNA transposable elements, transposition activities have only been directly demonstrated in the zebrafish Tzf element (Lam W L, Lee T S, Gilbert W. (1996) Proc Natl Acad Sci USA 93: 10870-10875) and in the medaka fish (Oryzias latipes) Tol2 element (Transposable element of Oryzias latipes, no. 2) (Koga A., Suzuki M., Inagaki H., Bessho Y. and Hori H. (1996) Transposable element in fish. Nature 383: 30).
The Tol1 element is a DNA transposable element present in medaka fish and at 100 to 200 copies per genome (Koga A., Sakaizumi M., Hori H. (2002) Zoolog Sci 19: 1 to 6 (Non-patent Document 1)). This element was discovered as an insert into a mutant tyrosinase gene. The mutant tyrosinge gene has a phenotype of a complete albino body color (Koga A., Inagaki H., Bessho Y., and Hori H. (1995) Mol Gen Genet. 249: 400-405. (Non-patent Document 2)). Tyrosinase is an enzyme essential in the biosynthesis of melanin. Tol2's transposition activity was demonstrated in Tol2 thereafter. Unlike Tol2, since excision and insertion was not directly detected in the Tol1 element, it was originally believed that the Tol1 element had already lost its transposition activity. In addition to the copy first discovered in a tyrosinase gene, other copies were also isolated and examined; however, none had the structure of a gene (Koga A., Inagaki H., Bessho Y., and Hori H. (1995) Mol Gen Genet. 249: 400-405. (Non-patent Document 2)). This further contributed to the belief that Tol1 had lost its transposition activity.
In 2001, a fish from an albino subline was found to have partial pigmentation, i.e., a mosaic pigmentation. Analysis of this fish, demonstrated that the Tol1 element had been excised from its insertion site (Tsutsumi M., Imai S., Kyono-Hamaguchi Y., Hamaguchi S., Koga A. and Hori H. (2006) Pigment Cell Res 19: 243-247. (Non-patent Document 3)). Since Tol1 had been excised, it is a DNA transposable element and that had not lost its transposition activity. However, its de novo insertion in a chromosome had not been observed. Furthermore, no transfer enzyme (transposase) has been found.
Transposable elements are used in genetic engineering and in molecular biology applications. For example, transposable elements are used to mutagenize genes, promoters, enhancers, etc., and are expected to be useful in gene therapies, and the like. The Tol2 element, which was discovered in medaka fish genomes, has already been used in such applications (Koga A., Hori H., and Sakaizumi M. (2002) Mar Biotechnol 4: 6-11. (Non-patent Document 4), Johnson Hamlet M. R., Yergeau D. A., Kuliyev E., Takeda M., Taira M., kawakami K., Mead P. E. (2006) Genesis 44: 438-445. (Non-patent Document 5), Choo B. G., Kondrichin I., Parinov S., Emelyanov A. Go W., Toh W. C. and Korzh V. (2006) BMC Dev Biol 6: 5. (Non-patent Document 6), Japanese Patent Application Laid-Open (JP-A) No. 2001-218588 (Patent Document 1)). In addition to the Tol2 element, the Sleeping Beauty element was artificially constructed from debris present in salmon genomes (Lvics Z., Hackett P. B., Plasterk R. H., Izsvak Z. (1997) Cell 91: 501-510. (Non-patent Document 7), National Publication of International Patent Application No. 2001-523450 (Patent Document 2)); the Frog Prince element was similarly constructed from a frog genome (Miskey C., Izsvak Z., Plasterk R. H., Ivics Z. (2003) Nucleic Acids res 31: 6873-6881. (Non-patent Document 8), National Publication of International Patent Application No. 2005-527216 (Patent Document 3)); and the piggyBac element was isolated from an insect genome (Wu S. C., Meir Y. J., Coates C. J., Handler A. M., Pelczar P., Moisyadi S, and Kaminski J. M. (2006) Proc Natl Acad Sci USA 103: 15008-15013. (Non-patent Document 9)); each of these elements has been used in gene introduction, etc. These elements are characterized by high transposition frequency. This characteristic is important when they are to be used in genetic engineering or molecular biology applications. Tol1 is estimated to have a high transposition ability given the large number of pigmented cells in mosaically-pigmented medaka fish.

Patent Document 1: JP-A No. 2001-218588
Patent Document 2: National Publication of International Patent Application No. 2001-523450
Patent Document 3: National Publication of International Patent Application No. 2005-527216
[Non-patent Document 1] Koga A., Sakaizumi M., Hori H. (2002) Zoolog Sci 19: 1-6.
[Non-patent Document 2] Koga A., Inagaki H., Bessho Y., Hori H. (1995) Mol Gen Genet. 249: 400-405.
[Non-patent Document 3] Tsutsumi M., Imai S., Kyono-Hamaguchi Y., Hamaguchi S., Koga A., Hori H. (2006) Pigment Cell Res Res 19: 243-247.
[Non-patent Document 4] Koga A., Hori H., Sakaizumi M. (2002) Mar Biotechnol 4: 6-11.
[Non-patent Document 5] Johnson Hamlet M. R., Yergeau D. A., Kuliyev E., Takeda M., Taira M., Kawakami K., Mead P. E. (2006) Genesis 44: 438-445.
[Non-patent Document 6] Choo B. G., Kondrichin I., Parinov S., Emelyanov A., Go W., Toh W. C., Korzh V. (2006) BMC Dev Biol 6: 5.
[Non-patent Document 7] Ivics Z., Hackett P. B., Plasterk R. H., Izsvak Z. (1997) Cell 91: 501-510.
[Non-patent Document 8] Miskey C., Izsvak Z., Plasterk R. H., Ivics Z. (2003) Nucleic Acids Res 31: 6873-6881.
[Non-patent Document 9] Wu S. C., Meir Y. J., Coates C. J., Handler A. M., Pelczar P., Moisyadi S., Kaminski J. M. (2006) Proc Natl Acad Sci USA 103: 15008-15013.

Problems to be Solved by the Invention

The Tol1 element is expected to be a novel transposase element. However, no full-length copy of the Tol1 element has yet been identified.
To use a DNA transposable element in genetic engineering or molecular biologic applications, a transposase, which transfers the transposable element, is required together with the element in a vector.
An object of the present invention is thus to provide a Tol1 element transposase to use Tol1 element in genetic engineering, etc. Another object of the present invention is uses for the Tol1 element (such as in a DNA introduction system or method).

Means for Solving the Problems

The present inventors tried to identify a transposase gene of Tol1 element using the above-described mosaically-pigmented fish (medaka fish). Database searches were repeatedly performed; from which a sequence was constructed which was estimated to be a nucleotide sequence of a transposase gene. Subsequently, the inventors identified a 2.9 kb cDNA, obtained from mRNA of the mosaically-pigmented fish. This cDNA included the coding sequence for a 851 amino acid peptide. The peptide encoded by this cDNA was found to cause transposition of Tol1 element in both human and mouse cells. Further, the peptide possessed a high transposition frequency which was comparable to that of Tol2. Thus, as a transposon, Tol1 element may have the same use and value, as a transposon, as the Tol2 element. The Tol1 element may also serve as an alternative to the Tol2 element. That is, the Tol1 element may effectively act on cell lines and organisms in which insufficient transposition frequencies is provided by the Tol2 element.
Further studies revealed that Tol1 and Tol2 do not induce mutual transposition (that is, a Tol1 element transposase does not induce transposition of Tol2, and vice versa). Thus, it would be possible to successively introduce two different DNAs to a target cell and by using both the Tol1 element and the Tol2 element. Further, after introducing the two different DNAs, providing a transposase corresponding to one of the elements will allow only one of the DNAs to be specifically transferred. Accordingly, the fact that Tol1 element and Tol2 element do not induce mutual transposition enhances usefulness of Tol1 element.
Further studies revealed the existence of Tol1 elements 18 kb and 20 kb in length. Thus, the Tol1 element may be useful for introducing (transposing) a large-sized DNA fragment. Two kinds of experiments were performed focusing on this point. First, an internal region, that is apparently unnecessary for transposition, was removed from the Tol1 element (Tol1-tyr, 1855 base pairs, SEQ ID NO: 10), which had been discovered as a fragment inserted in a tyrosinase gene. Thus, it was revealed that Tol1 element was efficiently transposed when at least 157 bp of the left end (5′ end region) and 106 bp of the right end (3′ end region) are present. Second, as described above, transposition efficiencies were measured for inserted DNA fragments of various sizes using the Tol1 element with a deleted internal region. It was found that the larger the DNA fragment to be inserted (i.e. as the distance increased between the left end and the right end of Tol1 element), the lower transposition frequency. However, even when the distance between the left end and the right end of Tol1 element was 22.1 kb, i.e., the largest-sized DNA fragment successfully tested, the transposition frequency was still significantly higher than a frequency of random incorporation into chromosomes when not employing transposition. Indeed, 22.1 kb is the longest DNA transposable element so far reported. The above studies, which were conducted by the present inventors, demonstrated that Tol1 1 element has excellent loading ability and is exceptionally useful for introducing (transposing) large DNA fragments.
Further studies revealed that excision of Tol1 element occurred also in Xenopus laevis—an important model organism of genetics and vertebrate development. The further studies suggested that Tol1 element functioned as a transposable element also in cells of Xenopus laevis. Thus, the Tol1 element may be highly versatile.
It was also confirmed that Tol1 element functioned in insects. First, a donor plasmid containing a nonautonomous copy of Tol1 element was injected into a fertilized silkworm egg together with RNA encoding a transfer enzyme of Tol1 element. The fertilized egg was kept warm to promote development, and plasmid DNA was then recovered from the embryo. Subsequently, the plasmid was analyzed by PCR. It was found that there were individual plasmids in which portions of Tol1 element had been excised. Further, genomic DNA from the embryo was analyzed by inverse PCR, which revealed that Tol1 element had been incorporated into chromosomes. As described above, both excision and insertion are two stages of a transfer reaction; both had occurred in the silkworm. This result has the following three meanings: (1) a transfer reaction of Tol1 element does not require an element from a host organism, or if the element is required, it is present in both protostomes (e.g., frogs and fish) and deuterostomes (e.g., insects); (2) systems of gene introduction, gene trapping, mutagenesis, and the like, can be performed in silkworms using Tol1 element; and (3) similar transposition systems can be constructed and used in wide varieties of animals.
As described above, a Tol1 element transposase was successfully identified and Tol1 element had preferable properties as a transposable element for use in genetic engineering. The present invention is based on such achievements and provides the following transposase, DNA introduction system, and the like.
[1] A Tol1 element transposase containing any of proteins selected from the group consisting of the following (a) to (c):
(a) a protein having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1;
(b) a protein having the amino acid sequence of SEQ ID NO: 2; and
(c) a protein having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 2, and having an enzymatic activity for transferring Tol1 element.
[2] A polynucleotide encoding a Tol1 element transposase containing any of nucleotide sequences selected from the group consisting of the following (a) to (c):
(a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2;
(b) the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4; and
(c) a nucleotide sequence homologous to the nucleotide sequence (b) and encoding a protein having an enzymatic activity for transferring Tol1 element.
[3] An expression construct containing the polynucleotide according to [2].
[4] The expression construct according to [3], further containing a promoter operably linked to the polynucleotide.
[5] The expression construct according to [3] or [4], further containing a poly-A additional signal sequence or a poly-A sequence connected to the polynucleotide at its downstream side.
[6] A DNA introduction system including:
(a) a donor plasmid having such a structure that a desired DNA is inserted in a transposase gene-defective Tol1 element; and
(b) a helper plasmid containing the transposase according to [1] or the polynucleotide according to [2].
[7] The DNA introduction system according to [6], wherein the Tol1 element has the inverted repeat sequence of SEQ ID NO: 5 in its 5′ end region and the inverted repeat sequence of SEQ ID NO: 6 in its 3′ end region.
[8] The DNA introduction system according to [6], wherein the Tol1 element contains DNA of the following (a) or (b):
(a) DNA having the nucleotide sequence of any of SEQ ID NOs: 10 to 12 or
(b) DNA having a nucleotide sequence homologous to the nucleotide sequence of any of SEQ ID NOs: 10 to 12, wherein a transposase having the amino acid sequence of SEQ ID NO: 1 binds to its end.
[9] The DNA introduction system according to [6], wherein the Tol1 element contains 5′ end side DNA and 3′ end side DNA obtained by deleting at least from the 158th base to the 1749th base counting from the 5′ end of the nucleotide sequence of SEQ ID NO: 10.
[10] The DNA introduction system according to [6], wherein the Tol1 element contains DNA having the nucleotide sequence of SEQ ID NO: 21 and DNA having the nucleotide sequence of SEQ ID NO: 22.
[11] The DNA introduction system according to any of [8] to [10], wherein a target site duplicated sequence is connected to the 5′ end and the 3′ end of the Tol1 element.
[12] The DNA introduction system according to [11], wherein the target site duplicated sequence contains the sequence of any of SEQ ID NOs: 13 to 15.
[13] The DNA introduction system according to any of [6] to [12], wherein the desired DNA is a gene.
[14] The DNA introduction system according to any of [6] to [13], wherein the donor plasmid is a vector obtained by inserting a desired DNA in a transposase gene-defective Tol1 element, and the helper plasmid is a vector containing the polynucleotide according to [2].
[15] The DNA introduction system according to [14], wherein the vector is a helper plasmid which contains a promoter operably linked to the polynucleotide.
[16] The DNA introduction system according to [14] or [15], wherein the vector is the helper plasmid which contains a poly-A additional signal sequence or a poly-A sequence connected to the polynucleotide at its downstream side.
[17] A DNA introduction method including a step of introducing the DNA introduction system according to any of [6] to [16] to a target cell which is a vertebrate cell.
[18] The DNA introduction method according to [17], wherein the target cell is a non-human vertebrate cell.
[19] The DNA introduction method according to [17] or [18], which includes a first step of introducing a first desired DNA to a target cell and further including a second step of introducing a second desired DNA to the target cell, with the second step using Tol2 element.
[20] A method of transferring a specific DNA into a genome, including a step of supplying a transposase corresponding to Tol1 element or Tol2 element to a cell obtained according to the method of [19].
[21] A method of transferring a specific DNA into a genome, including a step of introducing the transposase according to [1] or the polynucleotide according to [2] into a cell having a transposase gene-defective Tol1 element.
[22] The method according to [21], wherein another polynucleotide sequence is inserted in the Tol1 element.
[23] A cell obtained by the system according to any of [6] to [16], the method according to any of [17] to [19], or the method according to any of [20] to [22].
[24] A DNA introducing kit, including a donor plasmid made of an expression construct containing a transposase gene-defective Tol1 element and an insertion site and a helper plasmid including an expression construct containing the transposase according to [1] or the polynucleotide according to [2].
[25] The DNA introducing kit according to [24] wherein the Tol1 element has an insertion site between its 5′ end region DNA and its 3′ end region DNA which element is obtained by deleting DNA from at least the 158th base to the 1749th base counting from the 5′ end in the nucleotide sequence of SEQ ID NO: 10.
[26] The DNA introducing kit according to [24], wherein the Tol1 element has an insertion site between DNA having the nucleotide sequence of SEQ ID NO: 21 and DNA having the nucleotide sequence of SEQ ID NO: 22.
[27] The DNA introducing kit according to any of [24] to [26], wherein the insertion site includes a plurality of different kinds of restriction enzyme recognition sites.
[28] The DNA introducing kit according to any of [24] to [27], wherein the donor plasmid is a vector containing a transposase gene-defective Tol1 element and an insertion site, and the helper plasmid is a vector containing the polynucleotide according to [2].
[29] The DNA introducing kit according to [28], wherein the vector is the helper plasmid which contains a promoter operably linked to the polynucleotide.
[30] The DNA introducing kit according to [28] or [29], wherein the vector being the helper plasmid further contains a poly-A additional signal sequence or a poly-A sequence connected to the polynucleotide at its downstream side.
[31] A reconstructed transposon having a structure inserted with the polynucleotide according to [2] in a transposase gene-defective Tol1 element.
[32] The transposon according to [31], containing a promoter operably linked to the polynucleotide.
[33] The transposon according to [31] or [32], containing a poly-A additional signal sequence or a poly-A sequence connected to the polynucleotide at its downstream side.
[34] A DNA introduction system, containing the transposon according to any of [31] to [33].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mosaically-pigmented fish. Panels A to C show the same fish. The fish having one black eye and one red eye was photographed from its right side (panel A), its front (panel B), and its left side (panel C). The fish in Panel D is pigmented throughout its eyes and has a large number of pigmented dots on its back skin. The dots on its back are indicated with triangles. Panel E shows a first having spoke-form pigmentation in an eye. Panel F shows a peritoneum. A peritoneum is densely pigmented in wild-type fish and pigmentation absent from albino fish.

FIG. 2 shows structures of Tol1 nonautonomous and autonomous copies. Tol1-tyr is the first discovered Tol1 nonautonomous copy, and was inserted in a tyrosinase gene of fish A. Tol1-L1 is the first complete autonomous copy successfully identified, and contains a functional transposase gene. Genomic DNA of fish B was sheared, thereby producing fragments with 36 to 48 kb which were obtained and inserted into fosmid vector pCC1FOS to form a genomic library. This genomic library was screened to obtain Tol1-L1. An internal transposase gene (exon) is noted by a bar. An initiation codon (ATG) and a termination codon (TAG) of this gene are also shown. The underlined parts noted by “a” to “e” were used as targets for hybridization probes. “x” and “y” indicate positions of a 3′ RACE primer and a 5′ RACE primer, respectively. Sequences of these primers (primer x: SEQ ID NO: 16, primer y: SEQ ID NO: 17) correspond to positions of bases 152 to 181 and 457 to 332, respectively of the nucleotide sequence (SEQ ID NO: 3), which is registered in DDBJ/EMBL/GenBank as Accession No. AB264112.

FIG. 3 shows results of RACE. RNA was extracted from embryos 7 days after fertilization by fish A or fish B and synthesis of single stranded cDNA was performed using an oligo-dT primer. Then, 3′ RACE of this cDNA single strand was performed using primer “x” and a 3′ adaptor primer contained in a RACE kit. A DNA fragment was electrophoresed in a 1.0% agarose gel and transferred to a nylon membrane and then hybridized with probe “b”. The left panel shows the gel immediately after the electrophoresis and the right panel shows the gel after hybridization. Only one hybridization band was observed for cDNA from fish B. Subsequently, a portion of the gel corresponding to the hybridization band was excised and the DNA fragment contained therein was recovered. The recovered DNA fragment was ligated into a plasmid vector and a clone to which the probe bound was isolated by colony hybridization. 5′ RACE was performed only on RNA from fish B. Primer “y” and a 5′ adaptor primer contained in the RACE kit were used. Steps following PCR were the same as described above for 3′ RACE except probe “e” was used in hybridizations. One hybridization band was observed; the corresponding DNA fragment was isolated as described above.

FIG. 4 shows alignment using the Clustal-X program of a Tol1 element transposase and parts of other transposases of the hAT family. It is known that an element of the hAT family has some regions where amino acid sequences are preserved. The regions are expressed by A to F (Cited document 27). D and F out of A to F are located in a comparatively short region and are likely associated with dimerization of a protein. Elements having high homology to Tol1 were selected from the elements in the hAT family (Gen Bank Accession No. PF05699, at the World Wide Web (www) ncbi.nlm.nih.gov/Genbank/index.html). Results are shown in FIG. 4. Names encoded in UniProtKB were used as names of respective elements. Five letters indicating source species are added to the names. Positions of amino acids are indicated. A Clustal-X default method was used to color the amino acids.

FIG. 5 shows Southern blots including copies of Tol1 in medaka fish genomes. Genomic DNA was extracted from each one of fish A, fish B, HNI, or Hd-rR. HNI and Hd-rR are commonly-used inbred medaka fish lines. 8.0 μg of genomic DNA was prepared for each fish, completely digested with restriction enzyme PvuII, electrophoresed in a 1.0% agarose gel and transferred to a nylon membrane, which was then hybridized with probes “a” to “d” (see FIG. 2 for positions). Positions of molecular weight markers are shown. These results indicate that internal deletion is present in most copies of Tol1 present in medaka fish genomes.

FIG. 6 shows plasmids used for measurement of transposition frequency. Tol1-tyr (GenBank Accession No. D42062, SEQ ID NO: 10) was amplified from genomic DNA of fish A with 8 bp of adjacent TSD (CCTTTAGC (SEQ ID NO: 13)) and inserted into plasmid pUC19 to form a clone. Subsequently, a fragment of plasmid pCMV-Tag1 (bases 1675 to 3474 of the nucleotide sequence of GenBank Accession No. AF025668, SEQ ID NO: 18) was amplified by PCR and inserted into a SalI recognition site (bases 706 to 711 of the nucleotide sequence of GenBank Accession No. D42062) in Tol1-tyr. A neomycin-resistant gene is included in this fragment of pCMV-Tag1. The resulting plasmid was used as a donor plasmid. A helper plasmid was prepared by inserting bases 31 to 2817 (SEQ ID NO: 19) of Tol1 cDNA (nucleotide sequence of GenBank Accession No. AB264112, SEQ ID NO: 3) into a multicloning site of plasmid pCI. This multicloning site was present between a CMV promoter and a poly-A additional signal. A defective helper plasmid was prepared by introducing a mutation by PCR. Bases 996 to 1001 of the helper plasmid are ATGAAA, which corresponds to amino acids, methionine and lysine. Using PCR, ATGAAA was mutated to TAGTAA. This mutation resulted in sequential generation of two termination codons near the middle of a transposase's ORF. A filler plasmid was prepared by inserting 2.8 kb of a λDNA fragment into plasmid pCI instead of transposase cDNA.

FIG. 7 shows transposition of Tol1 in mammalian cells. A donor plasmid and a helper plasmid, a donor plasmid and a defective helper plasmid, or only a donor plasmid was transfected into HeLa cells or into NIH/3T3 cells. Filler plasmids were transfected as necessary. Selection for transfectants in G418 was then carried out. Pictures of 60 mm-dishes dyed with Giemsa stain solution are shown. A large number of G418-resistant colonies were generated only when a donor plasmid and a helper plasmid were transfected.

FIG. 8 shows nucleotide sequences of insertion points of inserted Tol1 copies. Genomic DNA was extracted from G418-resistant cells obtained by transfecting a donor plasmid and a helper plasmid and cut with EcoRI or PstI. The two restriction enzymes did not cut the donor plasmid. After genomic DNA was electrophoresed in a 1.0% agarose gel, DNA fragments with sizes from 3.7 to 9.0 kb were recovered from the gel, and ends thereof were ligated using T4 DNA ligase under a low DNA concentration (500 ng/2.0 ml) conditions. Inverse PCR was performed on the ligated DNA. The primer used recognized an end region of Tol1-tyr (the bases 162 to 133 (SEQ ID NO: 20) of the nucleotide sequence (SEQ ID NO: 10) of GenBank Accession No. D42062). When PCR products were electrophoresed, 10 or more bands were generated per reaction. The PCR product was inserted into a plasmid to form a clone and a nucleotide sequence thereof was examined using the same primer as used in the inverse PCR. nucleotide sequences around insertion points of these genomic DNA clones are shown. For reference, a sequence of a region corresponding to the donor plasmid is shown. 8 bp of TSD was observed in all insertion points.

FIG. 9 shows transposition frequencies of Tol1 and Tol2 in HeLa cells. Various amounts of a donor plasmid and a helper plasmid were combined, and transposition frequencies were measured for Tol1 (left panel) or for Tol2 (right panel). A donor plasmid of Tol2 was prepared based on a donor plasmid of Tol1. Specifically, the Tol1 left arm of the Tol1 donor plasmid was replaced with bases 1 to 755 of the Tol2 nucleotide sequence (GenBank Accession No. D84375, at the World Wide Web (www) ncbi.nlm.nih.gov/Genbank/index.html, SEQ ID NO: 9), and the right arm of the donor plasmid of Tol1 was replaced with bases 4147 to 4682 of the Tol2 nucleotide sequence. pHe103, as described in Cited document 33, was used as the helper plasmid of Tol2. The total amount of plasmid DNA was 1,000 ng in each experiment. Amounts of donor plasmid or helper plasmid are shown below the graphs. When the total amount of donor plasmid and helper plasmid was less than 1,000 ng, supplementary amounts of filler plasmid was added (the amount of filler plasmid used is omitted from the figure). Average values (±standard error) of the numbers of colonies found for three independent measurements are shown.

FIG. 10 shows mutual effects between Tol1 and Tol2 in HeLa cells. Plasmid DNAs were combined and transposition frequencies were measured. Six combinations were tested as described below the graph. In each experiment, the total amount of plasmid DNA was 1,000 ng. The amount of the filler plasmid used is omitted from the figure. Tol1 transposase and Tol2 transposase are capable of transferring only their own corresponding elements.

FIG. 11 shows the nucleotide sequence (DDBJ/EMBL/GenBank Accession No. AB264112, SEQ ID NO: 3) of cDNA (whole length 2,900 bp) of a Tol1 transposase and its deduced amino acid sequence (whole length 851 aa, SEQ ID NO: 2).

FIG. 12 shows a continuation of FIG. 11.

FIG. 13 shows a continuation of FIG. 12.

FIG. 14 shows variations in the length of naturally-occurring Tol1 elements. PCR was used to amplify an internal Tol1 portion from 130 clones whose genomic DNA hybridized with both Tol1 end regions. The primers used were 30 bp of Tol1's left end (1st to 30th bases) and 30 bp of its right end (1855th to 1826th bases). A bacteria colony was used as template. PCR conditions were as follows: [94° C., 2 minutes], 30×[94° C., 20 seconds; 64° C., 20 seconds; 72° C., 2 minutes], and [72° C., 5 minutes]. The PCR products were electrophoresed in a 1% agarose gel and lengths of the products were recorded. For clones in which amplification had not occurred using the above PCR conditions, PCR was performed with a longer elongation step (8 minutes instead of 2 minutes). Then, the PCR products were electrophoresed in a 0.8% agarose gel and the length of the products were recorded. In a third PCR condition, a 20 minute elongation step was used and the reaction products were electrophoresed in a 0.6% agarose gel. The lengths of 114 Tol1 elements were determined; their distributions are illustrated in the figure.

FIG. 15 shows a donor plasmid and a helper plasmid. FIG. 15 a shows a procedure for producing a short donor plasmid. pDon1855 is a plasmid containing the whole Tol1-tyr element and 8 bp of target site duplication (nucleotide sequence is CCTTTAGC), using pUC19 as a vector. PCR was performed using pDon1855 as a template to produce a short donor fragment. Outwardly-orientated primers were designed that recognized each Tol1 end region, the primers comprising a SalI recognition site in their 5′ ends. A PCR product was cut with SalI, and the both ends were ligated with the T4 DNA ligase into a circularized plasmid. A neomycin-resistant gene was inserted into a SalI site of the circularized plasmid. The neomycin-resistant gene was amplified from the pCMV-Tag1 plasmid (1675 to 3474 bases of DDBJ/EMBL/GenBank Accession No. AF025668) with a primer including a SalI recognition site. Black triangles shown in the drawing indicate end inverted repeat sequences, white triangles indicate target site duplications, and gray triangles indicate PCR primers. FIG. 15 b shows helper plasmids. pHe11851aa was a complete helper plasmid. pHe11851aa was produced by inserting the sequence encoding a Tol1 transfer enzyme (851 amino acids (SEQ ID NO: 2), 31 to 2817 bases of DDBJ/EMBL/GenBank Accession No. AB264112) between the CMV promoter of the pCI plasmid (Promega Corp., Madison, Wis., USA) and a poly-A additional signal. pHe1316aa is a defective helper plasmid. pHe1316aa was prepared by introducing a mutation into pHe1851aa by PCR. Bases 996 to 1001 of AB264112 is ATGAAA, which corresponds to amino acids, methionine and lysine, in a transfer enzyme. Using PCR, ATGAAA was mutated to TAGTAA. This mutation generated two termination codons near the middle of the ORF of the transfer enzyme.

FIG. 16 shows transposition frequencies of Tol1 elements having internal deletions. Only Tol1 arms are illustrated (not to scale) on the left of the figure. Plasmids containing these internally-deleted Tol1 elements were used as donor plasmids and transfected into cells along with the complete helper plasmid pHe1851aa (hatched rectangle) and the defective helper plasmid pHe1316aa (white rectangle). Transposition frequencies were measured. Average colony numbers from three measurements are shown in the graph. Horizontal lines in the graph are standard errors of the mean.

FIG. 17 shows donor plasmids having Tol1 elements of various lengths. pDon263Mcs (bottom panel) is an empty plasmid having a multicloning site. pDon263Mcs was prepared by removing each restriction enzyme recognition site present in pUC19, other than its HindIII recognition site. Six restriction enzyme recognition sites were then provided in a bonding portion of the Tol1 left and right arms, as shown in the drawing. The above-described modifications were made by PCR using a 5′-end modified primer. pDon263McsNeo (middle panel) was obtained by inserting a neomycin-resistant gene into a KpnI site and a PstI site of pDon263Mcs. Rectangles (upper panel) indicate DNA fragments ligated to form long Tol1 elements. These DNA fragments were produced by PCR amplification of various parts of bacteriophage λ (DDBJ/EMBL/GenBank Accession No. J02459). Numbers below the rectangles represent nucleotide positions of the amplified fragments. A PCR primer used for amplification included an EcoRI or HindIII recognition site in its 5′-end. The PCR product was cut with EcoRI or HindIII and then inserted into a respective site of pDon263McsNeo.

FIG. 18 shows transposition frequencies of long Tol1 elements. Transposition frequencies were measured for cells transfected with the complete helper plasmid pHe1851aa (hatched rectangle) or with the defective helper plasmid pHe1316aa (white rectangle). Donors are shown in the ExHy mode below the rectangles. Average colony numbers for three measurements are shown. Vertical lines indicate standard errors of the mean.

FIG. 19 shows nucleotide sequences around insertion points. Genomic DNA was extracted from a line derived from two colonies (N1 and N2) that had become neomycin-resistant and could be digested with HindIII. Since Tol1 element of a donor plasmid does not contain a HindIII recognition site, the Tol1 element itself should not be digested. The digested DNA was electrophoresed in a 0.8% agarose gel, and DNA fragments corresponding to 10 to 30 kb were recovered and ends were then ligated with T4 DNA ligase in a low concentration state (100 ng/500 μl). Inverse PCR was performed using the ligated DNA as template. The primers corresponded to the arms of Tol1 (130 to 101st bases and 1758 to 1787th bases of D42062). PCR conditions were as follows: [94° C., 2 minutes], 36×[94° C., 20 seconds; 64° C., 20 seconds; 72° C., 5 minutes], and [72° C., 5 minutes]. The PCR products were ligated into a plasmid, and the nucleotide sequence was amplified using the same primers as used in the inverse PCR. nucleotide sequences near insertion points are shown. nucleotide sequences of corresponding portions of the donor plasmid are aligned. An 8 bp target site duplication was formed in an insertion point of a host. The boxed portions correspond to PCR primer recognition sites used in later analyses of Tol1 elements (detail of which are described below in FIG. 20).

FIG. 20 shows an analysis of incorporated Tol1 element. FIG. 20 a shows amplification of Tol1 element. DNAs used as templates for PCR are pDon263McsNeoE20 (pDon) and two transformant lines (N1 or N2). The boxed portions in FIG. 19 correspond to PCR primer recognition sites used in these analyses. (P0 represents a sequence corresponding to plasmid pDon, P1 represents a sequence corresponding to cell line N1, and P2 represents a sequence corresponding to cell line N2). PCR was performed with the combination described above each electrophoresis lane. PCR conditions were as follows: [94° C., 2 minutes], 30×[98° C., 10 seconds; 68° C., 20 seconds], and [68° C., 10 minutes]. PCR products were electrophoresed in a 0.8% agarose gel with 2 μl of each 20 μl PCR product loaded per lane. A PCR product was generated only for a correct combination of a template and a primer. FIG. 20 b shows comparison of restriction enzyme digestion patterns. After ethanol precipitating the PCR products, precipitated PCR products were solubilized in sufficient distilled water to provide approximately the same final DNA concentration for each PCR product. The solubilized PCR products were cut with BamHI and KpnI, and electrophoresed in a 1.0% agarose gel. The whole region of the nucleotide sequence of pDon263McsNeoE20 is known. When pDon263McsNeoE20 is digested with BamHI and KpnI, five fragments from 1.5 kb to 11.7 kb are generated. A PCR product based on DNA from a transformant cell line had the same digestion pattern as the donor plasmid.

FIG. 21 shows structures of an indicator plasmid and a helper plasmid. plnd263GFP is an indicator plasmid that includes both ends of the first discovered Tol1 element (Tol1-tyr, 1855 bp, SEQ ID NO: 10) and a GFP gene inserted therebetween; plnd263GFP is formed by inserting the above into pUC19. pHe1851aa is a complete helper plasmid and was prepared by inserting a sequence encoding a Tol1 transfer enzyme (851 amino acids, SEQ ID NO: 2) between a CMV promoter of a pCI plasmid (Promega, Madison, Wis., USA) and poly-A additional signal. pHe1316aa is a defective helper plasmid that was prepared by introducing a mutation by PCR. Bases 996 to 1001 is ATGAAA, which corresponds to amino acids, methionine and lysine, in the transfer enzyme. Using PCR, the ATGAAA was mutated to TAGTAA so that two termination codons appeared near the middle of the transfer enzyme's ORF. In the drawing, abbreviations and nucleotide positions are included. The abbreviations are as follows: [Tol1] D84375 (Tol1-tyr element), [TPase] AB264112 (transfer enzyme gene), [pEGFP] U55763 (plasmid pEGFP-C1; Clontech Laboratories, Mountain View, Calif., USA). TSD shows target site duplication of the sequence CCTTTAGC. P_CMVrepresents a CMV promoter and PA represents a poly-A additional signal. The heavy line having a triangular right edge is an ORF contained in the complete and defective helper plasmids. The black triangles indicate end inverted repeat sequences present in the Tol1-tyr element. The open white triangles indicate positions and directions of PCR primers used to detect excision.

FIG. 22 shows detection by PCR of excision in an X. laevis embryo. A1 to A12 are 12 embryos from set A. B1 to B12 are 12 embryos from set B. The upper image shows electrophoresis bands of PCR products for PCR that amplified the whole region of Tol1 element. PCR conditions were as follows: [94° C., 120 seconds], 33×[94° C., 20 seconds; 64° C., 20 seconds; 68° C., 150 seconds], and [68° C., 60 seconds]. PCR products were electrophoresed in a 1.0% agarose gel with 5 μl of each 20 μl PCR product was loaded per lane. 2.4 kb bands were observed in all samples of A and B. The lower image shows results of PCRs that efficiently amplified a product of excision. PCR conditions were the same as in the upper image except the elongation step was shortened to 40 seconds. In addition, the agarose concentration in the electrophoresis was 2.0%. An approximately 535 bp band was observed in samples A1 to A12. No such band was observed in samples B1 to B12.

FIG. 23 shows nucleotide sequences around a breaking point of excision. “plnd” in the uppermost row is the sequence of plnd263GFP which is shown for reference. “TSD” indicates target site duplication. The full TSD is present in the plnd263GFP plasmid. Nucleotides enclosed within boxes are not present in plnd263GFP. Bracketed text, for example “[60 bp]”, represent sequences that are too long to be included in the figure. The sequences of corresponding portions are as follows: [60 bp], the 504th to 445th bases of L09137 (pUC19), and [30 bp], the 1821st to 1850th bases of D84375 (Tol-tyr).

FIG. 24 illustrates a phylogenetic tree of the animal world. The drawing illustrates a standard phylogenetic tree based on phylum or subphylum.

FIG. 25 illustrates the overall experiments performed herein, which diverge mid-course. The left branch illustrates detection of excision experiments and the right branch illustrates detection of insertion experiments.

FIG. 26 illustrates plasmids used as templates for RNA synthesis. The pTem851aa plasmid was prepared by inserting a sequence encoding a Tol1 transfer enzyme (nt 31 to 2817 of DDBJ/EMBL/GenBank Accession No. AB031079) into the pSP64 Poly (A) Vector (Promega corp.). “Pro” indicates a SP6 promoter and “An” indicates a poly (A) sequence. When RNA is synthesized using pTem851aa as a template, RNA (mRNA851aa) of about 2,900 nucleotides is transcribed. This RNA encodes a full-length transfer enzyme. The six bases (ATGAAA) encodes amino acids methionine and lysine. The pTem316aa plasmid was prepared by introducing a mutation, by PCR, which replaced the ATGAAA with two termination codons (TAGTAA). RNA (mRNA316aa) of about 2,900 nucleotides is formed; however the translated peptide ends at the two termination codons.

FIG. 27 shows a donor plasmid. White boxes identify a part of a tyrosinase gene (DDBJ/EMBL/GenBank Accession No. AB010101) of medaka fish. The black box identifies a Tol1 element (DDBJ/EMBL/GenBank Accession No. D84375). Triangles represent positions and directions of primers for detecting excision. nucleotide sequences are as follows: Pex1: 3,594 to 3,623 of AB010101; Pex2: 3,866 to 3,895 of AB010101; Pin1: 1,758 to 1,787 of D84375; and Pex2: 101 to 130 of D84375.

FIG. 28 shows PCR experiments for detecting excision. PCR was performed using a DNA template identified at the top of each lane. 10 pg of donor plasmid DNA was used in each PCR reaction; this is approximately the amount of DNA recovered from one embryo. Primers were Pex1 and Pex2. The upper image shows PCR products which confirm that the donor plasmid was recovered. PCR conditions were as follows: [94° C. for 120 seconds], 25×[94° C. for 20 seconds; 64° C. for 20 seconds; 72° C. for 150 seconds], and [72° C. for 150 seconds]. The lower image shows PCR products for detecting excision. PCR conditions were as follows: [94° C. for 120 seconds], 40×[94° C. for 20 seconds; 64° C. for 20 seconds; 72° C. for 20 seconds], and [72° C. for 20 seconds].

FIG. 29 shows nucleotide sequences of PCR products in excision detection. The “Donor” indicates a nucleotide sequence of both ends and portions subsequent thereto of Tol1 element on the donor plasmid. “Tol1” indicates Tol1 element, “Tyrosinase” indicates a portion derived from a tyrosinase gene of medaka fish, and “TSD” indicates target site duplication. “A1,” “A2,” and “A3” indicate PCR products for each of the three samples. Tol1 element regions were deleted in all three samples, and other sequences were contained in the regions. Lengths thereof are shown in the drawing and respective nucleotide sequences are shown in the bottom.

FIG. 30 shows nucleotide sequences of clones detected as having insertions. The “donor” indicates a nucleotide sequence of both ends and portions subsequent thereto of Tol1 element of the donor plasmid. “Clone 1” and “clone 2” indicate nucleotide sequences of corresponding portions of two clones obtained by an inverse PCR. A part of Tol1 element is expressed by outline characters on a black background.

BEST MODE FOR CARRYING OUT THE INVENTION

For the sake of simplicity, definitions and meanings of some terms used in the present specification are described below.
Inclusive expressions such as terms “contain” or “comprise” are used as expressions also including meanings such as “consist” or “is/are.”
A “nucleotide sequence encoding an amino acid sequence” in the present invention refers to a polynucleotide capable of encoding a protein. Therefore, a nucleotide sequence can have a coding region that corresponds to an amino acid sequence and also a sequence portion that does not correspond to the amino acid sequence. Further, degeneracy of a codon is also considered. Thus, in an “amino acid sequence encoded by a nucleotide sequence,” degeneracy of a codon is also considered.
The term “polynucleotide” refers to a polynucleotide in any form such as DNA and PNA (peptide nucleic acid), and RNA. The polynucleotide in the present invention is preferably DNA or mRNA.
The term “isolated” in the present invention is used interchangeably with “purified.” An “Isolated” transposase, which is derived from natural materials, will be substantially free of the natural materials other than the transposase. Such an isolated transposase is substantially free of impurities (i.e., natural materials other than the tranposase). For example, for a transposase isolated from natural materials, the content of impurities is less than about 20% by weight, preferably less than about 10%, more preferably less than about 5%, and further more preferably less than about 1%. An “isolated” transposase, which is prepared by genetic engineering, will be substantially free of other components derived from a host cell, culture medium, and the like. For example, for an isolated transposase prepared by genetic engineering, the content of impurities will be less than about 20% by weight, preferably less than about 10%, more preferably less than about 5%, and further more preferably less than about 1%. A “transposase” in the present specification can simply mean a “transposase in an isolated state” unless the context indicates that a different meaning is intended. The term “enzyme” can be used in place of the term “transposase”.
An “Isolated” polynucleotide, which is derived from natural materials, is separated from other nucleic acids coexisting in the natural materials. However, other nucleic acid components such as a flanking sequence in a natural state (for example, a sequence of a promoter region, and a terminator sequence) may be present. For example, “isolated” genomic DNA, substantially lacks other DNA components naturally coexisting with the genomic DNA. An “isolated” DNA prepared by genetic engineering (such as cDNA), will be substantially free of cell components, culture solutions, and the like. An “isolated” DNA, which is prepared by chemical synthesis, will be substantially free of precursors (i.e., raw materials) such as dNTP, chemical substances used in a synthesis process, and the like. A “polynucleotide” in the present specification can simply mean a polynucleotide in an isolated state unless the context indicates that a different meaning is intended.
The term “DNA introduction” in the present specification means to introduce DNA into a target cell. Accordingly, genetic modifications (such as mutagenesis and gene targeting) are also included in the concept of DNA introduction. (Tol1 element transposase)
A first aspect of the present invention provides a Tol1 element transposase, which is based on Applicants' discovery of a Tol1 element transposase. A “Tol1 element transposase” refers to an enzyme capable of transferring Tol1 element, a transposon as found in medaka fish. Hereinafter, the term “transposase”, unless otherwise specified, refers to the “Tol1 element transposase”.
In one embodiment, the transposase of the present invention has an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1. As shown in examples described later, the nucleotide sequence (including a termination codon) is an ORF (open reading frame) encoding a Tol1 element transposase. SEQ ID NO: 2 (851 amino acids) is a deduced amino acid sequence encoded by the ORF. Another embodiment of the present invention is a protein having the amino acid sequence of SEQ ID NO: 2 (FIGS. 11 to 13). Note that a cDNA corresponding to the amino acid sequence (including poly-A, FIGS. 11 to 13, SEQ ID NO: 3) is registered in DDBJ/EMBL/GenBank as Accession No. AB264112 (not published as of Dec. 13, 2006).
A transposase of the present invention has high specificity to a nucleotide sequence of substrate DNA. It does not have substantial enzymatic activity for transferring Tol2 element, which is a transposon also found in medaka fish.
In general, when a part of an amino acid sequence of a protein is modified, the resulting protein may have equivalent activity as an unmodified protein. That is, modification of an amino acid sequence may not have a substantial effect on a protein's activity; the protein's activity may remain after modification. Thus, another aspect of the present invention provides a protein having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 2 and having an enzymatic activity for transferring Tol1 element (hereinafter referred to as a “homologous protein”). The “homologous amino acid sequence” herein refers to an amino acid sequence different from of SEQ ID NO: 2, however, the “homologous protein” substantially retains the enzymatic activity for transferring Tol1 element as the amino acid sequence of SEQ ID NO: 2.
The “partial difference of an amino acid sequence” means a change in an amino acid sequence, e.g., by deletion or substitution, of one to several amino acids in an amino acid sequence, or addition or insertion of one to several amino acids, or combinations thereof. The difference of an amino acid sequence herein is acceptable as long as enzymatic activity for transferring Tol1 element is retained (some fluctuation in activity is acceptable). As long as this requirement is met, a location at which an amino acid sequence is different is not particularly limited, and the difference may be generated in a plurality of locations. “Plurality” herein is, for example, less than about 30% of the amino acids, preferably it is less than about 20%, more preferably it is less than about 10%, further more preferably it is less than about 5%, and most preferably it is less than about 1%. That is, a homologous protein has an identity of, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, further more preferably about 95% or more, and most preferably about 99% or more relative to the amino acid sequence of SEQ ID NO: 2.
It is preferable that a homologous protein has amino acid substitutions in an amino acid residue that is not essential to an enzymatic activity for transferring Tol1 element. A “preservative amino acid substitution” herein refers to substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families by their side chains, such as basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Preservative amino acid substitution is preferably a substitution of amino acid residues in the same family.
A percent identity of two amino acid sequences or two nucleotide sequences (hereinafter, “two sequences”) can be determined by the following procedure, for example. First, two sequences are aligned so that they can be optimally compared (for example, a gap may be introduced in a first sequence to optimize alignment with a second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as a molecule at a corresponding position thereto in the second sequence, the molecules are considered identical. A percent identity of two sequences is a function of the number of identical positions between the two sequences (i.e., percent identity=number of identical positions/total number of positions×100). The number and sizes of gaps required for alignment are also taken into consideration.
Mathematical algorithms can be used to compare two sequences and determine their percent identity. Specific examples of the mathematical algorithm applicable to comparison of sequences include an algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77, but are not limited thereto. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain an amino acid sequence homologous to a specific amino acid sequence, for example, a BLAST polypeptide search may be carried out by the XBLAST program at a score of 50 and a wordlength of 3. To obtain a nucleotide sequence homologous to a specific nucleotide sequence, for example, BLAST nucleotide search may be carried out by the NBLAST program at a score of 100 and a wordlength of 12. To obtain a gap alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 is used. When BLAST and Gapped BLAST are employed, a default parameter of a corresponding program (such as XBLAST and NBLAST) can be used. See the World Wide Web (www) ncbi.nlm.nih.gov for details. Examples of another mathematical algorithm applicable to comparison of sequences include the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an algorithm is incorporated in the ALIGN program available from, for example, the GENESTREAM network server (IGH Montpellier, France) or the ISREC server. When the ALIGN program is used for comparison of amino acid sequences, for example, the PAM120 residue mass table is used, a gap length penalty can be set to 12 and a gap penalty can be set to 4.
An identity of two amino acid sequences can be determined with the GAP program of the GCG software package using Blossom 62 matrix or PAM250 matrix, and by setting a gap load to 12, 10, 8, 6, or 4 and a gap length load to 2, 3, or 4. Further, a homology of two nucleic acid sequences can be determined with the GAP program of the GCG software package (available from the World Wide Web (www) gcg.com) by setting a gap load to 50 and a gap length load to 3.
The transposase of the present invention can be prepared by genetic engineering. For example, a suitable host cell (such as Bacillus coli) is transformed with a polynucleotide encoding the transposase of the present invention thereby creating a transformant. A protein expressed in the transformant which synthesizes the transposase is recovered. The recovered protein is purified in a manner depending on its further applications. Various modifications of a recombinant transposase are possible. For example, DNA encoding the transposase and another suitable DNA can be inserted into the same vector. Thereby, a recombinant protein is produced from the vector, which comprises the transposase connected to any peptide or protein encoded by the other suitable DNA. Further, the transposase can be modified by addition of a sugar chain and/or lipid, or modified to generate processing of N ends or C ends. Due to the above-described modifications, extraction of a recombinant protein, simplification of purification, addition of biological functions, and the like are possible.
The methods for preparing the transposase of the present invention is not limited to genetic engineering. For example, if natural materials exist, the transposase can also be prepared from the natural materials by a standard techniques (e.g., fractionation, extraction, purification, etc.). The transposase of the present invention is generally prepared in an isolated state.

(Polynucleotide Encoding Tol1 Element Transposase)

A second aspect of the present invention provides a polynucleotide encoding the transposase. In one embodiment, the polynucleotide is made of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2. Specific examples of the nucleotide sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4. The nucleotide sequence of SEQ ID NO: 1 is an ORF encoding a Tol1 element transposase. Further, the nucleotide sequence of SEQ ID NO: 3 corresponds to a full-length cDNA encoding a Tol1 element transposase. The nucleotide sequence of SEQ ID NO: 4 corresponds to a genomic DNA sequence (4355 base pairs, absent a target site duplicated sequence (TSD)) which encodes the mRNA corresponding to the full-length cDNA.
If part of a polynucleotide encoding a protein is modified, the resulting protein may have equivalent enzymatic activity as the protein encoded by an unmodified polynucleotide. That is, modification of a nucleotide sequence may not have a substantial effect on the encoded modified protein's enzymatic activity, and the unmodified protein's activity may be retained in the modified protein. Thus, another embodiment of the present invention provides a polynucleotide made of a nucleotide sequence homologous to the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4, and encoding a protein having an enzymatic activity for transferring Tol1 element (hereinafter a “homologous polynucleotide”). The “homologous nucleotide sequence” herein refers to a nucleotide sequence different from the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, in which the difference, however gives no substantial effect on a function of the protein encoded by the homologous nucleotide sequence (function meaning enzymatic activity for transferring Tol1 element).
Specific examples of a homologous polynucleotide include a polynucleotide that hybridizes (under stringent conditions) to a polynucleotide made of a nucleotide sequence complementary to the nucleotide sequences of any of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4 when under stringent conditions. The “stringent conditions” herein refers to conditions where a so-called specific hybrid is formed, but a non-specific hybrid is not formed. Such stringent conditions are known to a skilled person, and are described elsewhere, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). The stringent conditions include, for example, such conditions that incubation is carried out at about 42° C. to about 50° C. using a hybridization solution (50% formamide, 10×SSC (0.15 MNaCl, 15 mM sodium citrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml of modified salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)), and then washing is carried out at about 65° C. to about 70° C. with 0.1×SSC and 0.1% SDS. Preferable stringent conditions include, for example, such conditions that 50% formamide, 5×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml of modified salmon sperm DNA, 50 mM phosphate buffer (pH 7.5) is used as a hybridization solution.
Other specific examples of a homologous polynucleotide include a polynucleotide made of a nucleotide sequence containing a substitution, deletion, insertion, addition, or inversion at one or a plurality of positions when compared to the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, and encoding a protein having an enzymatic activity for transferring Tol1 element. Substitution and deletion of bases may occur in a plurality of positions. “Plurality” herein indicates, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases. The number of bases capable of being changed depends the specific positions of the bases and the type of amino acid encoded by the polynucleotide. Such a homologous polynucleotide as described above can be obtained by modifying a polynucleotide having the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4 by substitution, deletion, insertion, addition and/or inversion; such modifications can be via a restriction enzyme digestion, digestion with exonuclease, treatment with DNA ligase, or the like; via a site-specific mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York); and random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). A homologous polynucleotide can also be obtained by other methods such as exposure to ultraviolet radiation.
Other examples of a homologous polynucleotide include a polynucleotide in which such difference in bases, as described above, are recognized due to a polymorphism typified by SNP (monobasic polymorphism).
An isolated polynucleotide of the present invention can be prepared using a standard genetic engineering technique, molecular biology technique, biochemical technique, or the like, thereby preparing a sequence as disclosed in the present specification or attached sequence listings. Specifically, the polynucleotide of the present invention can be isolated from a medaka fish (Oryzias laptipes) genomic DNA library or cDNA library or a cell extract of medaka fish. In each, isolation can be performed using an oligo-nucleotide probe/primer capable of specifically hybridizing to the polynucleotide of the present invention. The oligo-nucleotide probe/primer can be synthesized using a commercially-available automated DNA synthesizer. Preparation of such libraries are taught in Molecular Cloning, (Third edition, Cold Spring Harbor Laboratory Press, New York).
For example, a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 can be isolated from a medaka fish cDNA library using a probe, which hybridizes to the whole or a part of the nucleotide sequence or a sequence complementary thereto. Also, the polynucleotide can be amplified and isolated using a nucleic acid amplification reaction (for example, PCR) using a synthesized oligo-nucleotide primer designed to specifically hybridize to a part of the nucleotide sequence.

(Expression Construct Containing Tol1 Element Transposase)

A further aspect of the present invention relates to an expression construct containing the polynucleotide of the present invention. A promoter is preferably incorporated in the expression construct of the present invention. However, when a polynucleotide contained in the expression construct already has a promoter region, a second promoter can be omitted.
The promoter is operably linked to the polynucleotide of the present invention. In an expression construct comprising a polynucleotide that is operably-linked to a promoter, the promoter enables expression of the polynucleotide in a target cell. Herein, “a promoter that is operably linked to a specific polynucleotide sequence” has the same meaning as “a specific polynucleotide sequence under control of a promoter,” and generally, a specific polynucleotide sequence is linked to the 3′-end side of the promoter directly or via another sequence.
CMV-IE (cytomegalovirus initial gene-derived promoter), SV40ori, retrovirus LTP, SRα, EF1α, β-actin, and like promoters can be used. Mammal-tissue-specific promoters such as an acetylcholine receptor promoter, an enolase promoter, an L7 promoter, a nestin promoter, an albumin promoter, an alpha-fetoprotein promoter, a keratin promoter, and an insulin promoter may be used.
In the expression construct of the present invention, a poly-A additional signal sequence, a poly-A sequence, an enhancer sequence, a selective marker sequence, and the like can also be included. Stability of mRNA generated from the expression construct is improved by use of a poly-A additional signal sequence or a poly-A sequence. The poly-A additional signal sequence or the poly-A sequence is connected downstream to the polynucleotide. Improvement of expression efficiency occurs with use of an enhancer sequence. Further, when an expression construct containing a selective marker sequence is used, presence or absence (and a degree thereof) of the expression construct can be confirmed using the selective marker.
Preparing an expression construct by including a promoter, the polynucleotide sequence of the present invention, an enhancer sequence (if necessary), and a selective marker sequence (if necessary) can be performed by standard recombinant DNA techniques, e.g., using a restriction enzyme and a DNA ligase (See, e.g., Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York can be referred to).
The expression construct of the present invention can introduce the polynucleotide sequence of the present invention into a target cell. Although the type of expression construct used is not particularly limited, the expression construct preferably is an expression vector. The “expression vector” herein refers to a nucleic acid molecule capable of introducing a contained polynucleotide into a desired cell (target cell) and of expressing the polynucleotide in the cell. Examples of such include a viral vector and a non-viral vector. A gene introduction method using a viral vector occurs via infection of a cell with a virus, and a high gene introduction efficiency can be obtained. An adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a herpesvirus vector, a Sendai virus vector, and the like, have been developed for use in the present invention. For an adeno-associated virus vector, a retrovirus vector, and a lentivirus vector, foreign genes included into a vector are incorporated into a host chromosome, and stable, long-term expression can be expected. Since retrovirus vectors require cell division for incorporation of a foreign gene into a host chromosome, a retrovirus vector is not appropriate for gene introduction into a non-dividing cell. On the other hand, lentivirus vectors and adeno-associated virus vectors cause incorporation of foreign genes into host chromosomes after infection and in non-dividing cells; therefore, these vectors are effective for expressing foreign genes stably and for a long time in non-dividing cells, such as nerve cells and liver cells.
Each viral vector can be prepared by following standard methods or using a commercially-available kit. For example, preparation of an adenovirus vector can be carried out by a COS-TPC method or a full-length DNA introduction method. The COS-TPC method produces a recombinant adenovirus by co-transfecting a recombinant cosmid containing a desired cDNA or an expression cassette and a parent virus DNA-terminal protein complex (DNA-TPC) into 293 cells; then homologous recombination in the 293 cells integrates foreign genes into the 293 cell's chromosome (Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). Full-length DNA introduction method produces a single recombinant adenovirus using restriction digestion of a recombinant cosmid comprising a desired gene; thereafter transfecting the product to 293 cells (Miho Terashima, Saki Kondo, Yumi Kanegae, and Izumi Saito (2003) Experimental Medicine 21 (7) 931). The COS-TPC method can be performed with the Adenovirus Expression Vector Kit (Dual Version) (TAKARA BIO INC.) and Adenovirus genome DNA-TPC (TAKARA BIO INC.). Further, the full-length DNA introduction method can be performed with Adenovirus Expression Vector Kit (Dual Version) (TAKARA BIO INC.).
A retrovirus vector can also be prepared by the following procedure. First, viral genes (e.g., gag, pol, and env genes) other than packaging signal sequences between LTR (Long Terminal Repeat) present on the both ends of a virus genome are removed. Then a desired gene is inserted therein. The constructed viral DNA is introduced in a packaging cell constitutionally expressing gag, pol, and env genes. Here, only a vector RNA having a packaging signal sequence is incorporated in a virus particle and a retrovirus vector is produced.
As a vector obtained by application or improvement in an adeno vector, a vector in which specificity is improved by modification of a fiber protein (specific infection vector), and a gutted vector from which improvement in an expression efficiency of a desired gene can be expected (helper-dependent vector), and the like, have been developed. The expression vector of the present invention can be constructed as such a viral vector.
As a non-viral vector, a liposome, a positively charged liposome (Felgner, P. L., Gadek, T. R., Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), HVJ (Hamagglutinating virus of Japan)-liposome (Dzau, V. J., Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci., 93:11421-11425, 1996, Kaneda, Y., Saeki, Y. & Morishita, R., Molecular Med. Today, 5:298-303, 1999), and the like have been developed. The expression vector of the present invention can be constructed as such a non-viral vector.

(DNA Introduction System Utilizing Tol1 Element)

Another aspect of the present invention relates to a DNA introduction system using Tol1 element. The DNA introduction system of the present invention can be used for introducing specific DNA into a target cell. In other words, specific DNA can be introduced into genomic DNA of a target cell using the DNA introduction system of the present invention. The DNA introduction system of the present invention is used for genetic manipulation, such as gene introduction and gene modification.
The DNA introduction system of the present invention includes a donor plasmid and a helper plasmid. The donor plasmid and the helper plasmid preferably exist as distinct plasmids. That is, it is preferable that the donor plasmid and the helper plasmid are physically separate. However, the donor plasmid and the helper plasmid may coexist in a single plasmid.
The donor plasmid delivers a desired DNA to a target cell, with the desired DNA inserted in a transposase gene-defective Tol1 element.
The “target cell” means a cell to which the DNA introduction system of the present invention is applied, that is, a cell to be genetically manipulated using the DNA introduction system of the present invention. The “target cell” herein indicates a vertebrate cell, as examples cells of mammals (e.g., human, monkey, cattle, horse, rabbit, mouse, rat, guinea pig, and hamster), birds (e.g., chicken and quail), fish (e.g., medaka and zebrafish), and amphibians (e.g., frog). Examples of target cells include: a myocardial cell, a smooth muscle cell, an adipose cell, a fibrocyte, a bone cell, a chondrocyte, an osteoclast, a parenchymal cell, an epidermal keratinocyte (keratinocyte), epithelial cells (e.g., skin epidermal cell, corneal epithelial cell, conjunctival epithelial cell, oral mucosal epithelium, follicle epithelial cell, oral mucosal epithelial cell, airway mucosal epithelial cell, and intestinal mucosal epithelial cell), endothelial cells (e.g., corneal endothelial cell and vascular endothelial cell), a nerve cell, a glial cell, a splenic cell, a pancreatic β cell, a mesangium cell, a Langerhans cell, a liver cell, or precursor cells thereof, or mesenchymal stem cells (MSC), embryonic stem cells (ES cells), embryonic germ cells (EG cells), adult stem cells, fertilized eggs, and the like. Cells can have abnormalities, including cancer cells, or established cell lines such as HeLa cells, CHO cells, Vero cells, HEK293 cells, HepG2 cells, COS-7 cells, NIH3T3 cells, Sf9 cells, and the like.
The DNA introduction system of the present invention can be used on an isolated target cell or to a target cell within an organism. Therefore, the present invention can be carried out in vitro, in vivo, or ex vivo. “Isolated” used herein refers to a state of being taken out from the original environment thereof (for example, from an organism). Accordingly, an isolated target cell exists in a culturing vessel or another container, and the cell can be artificially manipulated in vitro. Specifically, a cell separated from a living body and cultured ex vivo (including an established cell line) can be an isolated target cell. As long as a target cell is “isolated” according to the above-described meaning, the target cell is an isolated cell even if within an organism.
The “isolated target cell” can be isolated from a biological organism. Isolated target cells can also be obtained from RIKEN BioResource Center (independent administrative institution), the National Institute of Technology and Evaluation Institute (independent administrative institution), ATCC (American Type Culture Collection), DSMZ (German Collection of Microorganisms and Cell Cultures), and the like.
In one embodiment of the present invention, the DNA introduction system of the invention is applied to vertebrate cells outside a human. That is, the DNA introduction system is carried out on a target cell isolated from a human or a non-human vertebrate.
Tol1 element used in the donor plasmid is a transposase gene-defective Tol1 element. “transposase gene-defective” means containing no functional transposase gene, and also includes situations when some of the transposase gene remains as long as the transposase gene cannot be expressed as a functioning enzyme; thus, it is not limited to when the transposase gene is completely deleted. In other words, a part of the transposase gene's sequence is changed such that the unchanged part of the gene has lost its activity. Such a state is included in the meaning of “transposase gene-defective.”
Tol1 element is a DNA-type element existing in about 100 to 200 copies in a genome of medaka fish (Koga A., Sakaizumi M., and Hori H. (2002) Zoolog Sci 19: 1-6. (Cited document 10)). It was discovered as a fragment inserted in a tyrosinase gene (Koga A., Inagaki H., Bessho Y., and Hori H. (1995) Mol Gen Genet. 249: 400-405. (Cited document 11)). The sequence (SEQ ID NO: 10) of this fragment (Tol1-tyr, 1855 base pairs, lacking a target site duplicated sequence (TSD)) is registered in GenBank as Accession No. D42062. The inverted repeat sequence, characteristic of Tol1 element, was identified from an analysis of Tol1-tyr (Koga A., Sakaizumi M., and Hori H. (2002) Zoolog Sci 19: 1-6. (Cited document 10)). A preferable embodiment of the present invention uses a Tol1 element having the inverted repeat sequence of SEQ ID NO: 5 in its 5′ end region and the inverted repeat sequence of SEQ ID NO: 6 in its 3′ end region. That is, in a Tol1 element in this embodiment, the sequence 5′-cagtagcggttcta-3′ (SEQ ID NO: 5) is present in the 5′ end region of its sense strand and the sequence 5′-tagaaccgccactg-3′ (SEQ ID NO: 6) is present in the 3′ end region of its sense strand. In addition, all Tol1 elements reported so far, including Tol1-tyr, have defective transposase genes, and are envisioned as being a Tol1 element in the present invention. As specific examples of Tol1 element that can be used in the present invention, nucleotide sequences thereof are shown in SEQ ID NOs: 10 to 12. Note that the nucleotide sequence of SEQ ID NO: 11 is a cloned sequence (969 base pairs) obtained by removing an internal region of 886 base pairs from Tol1-tyr; it is transposable similar to Tol1-tyr. Further, the nucleotide sequence of SEQ ID NO: 12 is a cloned sequence (297 base pairs) obtained by removing an internal region of 1576 base pairs from Tol1-tyr and adding recognition sites for six restriction enzymes. These added restriction enzyme recognition sites allow insertion of other DNA fragments; the nucleotide sequence of SEQ ID NO: 12 is transposable similar to Tol1-tyr.
A modified element of any of these examples can also be used. The “modified element” herein indicates a polynucleotide molecule made of a nucleotide sequence homologous to the nucleotide sequence of any of SEQ ID NOs: 10 to 12; it functions as a transposon similar to the unmodified polynucleotide molecule. A transposase having the amino acid sequence of SEQ ID NO: 2 can bond to an end of the modified element. The term “homologous,” is defined below in the section of the “polynucleotide encoding a Tol1 element transposase”.
As shown in examples described below, the present inventors found that transposition efficiency was not lost when an internal region of 1592 base pairs (from its 158th base to 1749th base counting from the 5′-end) were deleted from Tol1-tyr. Based on this finding, one embodiment of the present invention uses Tol1 element made of 5′-end region DNA and 3′-end region DNA, which is obtained by deleting at least bases from 158th base to 1749th base (1592 base pairs) counting from the 5′-end in the nucleotide sequence of Tol1-tyr (the nucleotide sequence of SEQ ID NO: 10). In other words, the embodiment uses a donor plasmid having a structure in which a desired DNA is inserted between DNA of the 5′ end region of the nucleotide sequence of SEQ ID NO: 10 (157 base pairs at maximum length) and DNA of the 3′ end region of the nucleotide sequence of SEQ ID NO: 10 (106 base pairs at maximum length). Maximizing the amount of a loadable foreign DNA is obtained by deleting as much as possible of the internal region that is unnecessary for transposition activity. It was suggested that, when 1592 base pairs in the internal region of Tol1-tyr is deleted, as described above, greater than 20 kb of foreign DNA can be introduced (examples of this are described below). A specific example of the 5′-end region DNA is DNA having the nucleotide sequence of SEQ ID NO: 21 (157 base pairs), and a specific example of the 3′-end region DNA is DNA having the nucleotide sequence of SEQ ID NO: 22 (106 base pairs).
Tol1 element can be easily prepared by PCR, and the like, with a Tol1 element-specific primer (see examples described below) using medaka fish genomic DNA as template. Regarding details of preparation methods, refer to examples described below, or Koga A., Sakaizumi M., and Hori H. (2002) Zoolog Sci 19: 1-6, Tsutsumi M., Imai S., Kyono-Hamaguchi Y., Hamaguchi S., Koga A., and Hori H. (2006) Pigment Cell Res 19: 243-247, and the like.
“A desired DNA” contained in a donor plasmid refers to DNA introduced into genomic DNA of a target cell by the DNA introduction system of the present invention. The DNA introduction system of the present invention can be useful for gene introduction for the purposes of functional analysis of genes, improvement and restoration (treatment) of specific functions, addition of new traits, differentiation induction, production of useful proteins (such as interferon, insulin, erythropoietin, and antibodies), formation of transgenic animals, and the like. When the DNA introduction system of the present invention is used, specific genes are the “desired DNA.” Examples of the genes herein can include genetic disease-associated genes such as an adenosine deaminase (ADA) gene, a factor IX gene, a granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, a p53 cancer suppressing gene, a simple herpes virus thymidine kinase (HSV-tk) gene, a vascular endothelial growth factor (VEGF) gene, and a hepatocellular growth factor (HGF), genes encoding hormones such as insulin and erythropoietin, genes encoding growth factors such as interferon, an insulin-like growth factor, an epidermal growth factor (EGF), a fibrocyte growth factor (FGF), and interleukins, genes encoding antibodies (for therapy, diagnosis, detection, etc.), marker genes such as a green fluorescent protein (GFP) gene, a β-galactosidase (lacZ) gene, a chloramphenicol-resistant (CAT) gene, and a luciferase (LUC) gene, and genes having unknown functions. In addition to genes existing in nature, genes obtained through artificial manipulation (artificial genes) can also be used. Further, genes may be normally expressed in the same cell type or in a different cell type as a target cell. Desired DNA encoding two or more genes may be introduced.
When genetic modification is intended, any DNA capable of destroying or inactivating a function in a target cell, for example, a modified gene of the target gene, is used as the “desired DNA.”
An insertion position of the “desired DNA” is not particularly limited as long as the function of a transposon of Tol1 element (transposition function) remains unaffected. That is, the “desired DNA” may be inserted in a position other than in either both end that are acting sites of a transposase. For example, endogenous restriction enzyme recognition sites (e.g., SalI) existing in the region other than both ends may be used as an insertion site in Tol1 element. Alternatively, an insertion site may be artificially formed without using an endogenous restriction enzyme recognition site.
In one preferable embodiment of the present invention, a target site duplicated sequence is connected to the 5′-end and the 3′-end of Tol1 element. The “target site duplicated sequence,” that is, TSD (target site duplication), indicates a tandem repeat sequence formed in transposition. When a transposon is inserted, since double stranded DNA is broken at different positions, the sequence between them is duplicated, resulting in formation of a TSD. In the case of Tol1 element, TDS with 8 bp in one side is formed. For example, a TSD having the sequence of any of SEQ ID NOs: 13 to 15 can be used in the present invention. In addition, the sequences of SEQ ID NOs: 13, 14, and 15 correspond to TSD of Tol1-tyr, TSD of Tol1-L1, and TSD of Tol1-L2, respectively.
To achieve high introduction efficiency, a vector in which desired DNA, Tol1 element, etc., is incorporated therein as an expression cassette is included in a donor plasmid. The kind of vector used is not particularly limited. Regarding kinds, production methods, and the like of the vector, the above description (section of the expression construct of the present invention) can be referred to.
The helper plasmid delivers a transposase into a target cell, and includes the transposase of the invention (i.e., Tol1 element transposase) or the polynucleotide of the invention (i.e., polynucleotide encoding the Tol1 element transposase). When the DNA introduction system of the present invention is introduced into the target cell, a transposase supplied by the helper plasmid acts on Tol1 element supplied by the donor plasmid. As a result, the desired DNA inserted in the Tol1 element is incorporated in the genomic DNA of the target cell.
To achieve a high introduction efficiency, the helper plasmid is preferably also constructed as a vector similar to the donor plasmid. That is, it is preferable to use a vector in which an expression cassette containing a polynucleotide encoding a Tol1 element transposase is incorporated in the helper plasmid.
Introduction of the donor plasmid and the helper plasmid into a target cell can be carried out by a calcium phosphate co-precipitation method, lipofection (Felgner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1984)), an HVJ liposome method, a DEAF dextran method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165 (1984)), microinjection (Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci. U.S.A. 73, 366-370 (1976)), a gene gun method, an ultrasonic gene introduction method, etc., in consideration of kinds of the target cell, forms of the donor plasmid and the helper plasmid, and the like. When a viral vector is used as an expression construct, introduction into the target cell is performed by infection.
The donor plasmid and the helper plasmid are not necessarily introduced into a target cell simultaneously. However, it could be preferable that the both plasmids are simultaneously co-introduced into the target cell from the viewpoints of operationality and mutual actions of the both plasmids.
The present invention provides further uses of the DNA introduction system of the invention. One use is a DNA introduction method. In the DNA introduction method of the present invention, a step of introducing the DNA introduction system of the invention is carried out on a vertebrate target cell. Further, based on the finding that Tol1 element and Tol2 element do not influence each other's transposition, provided is a DNA introduction method characterized by a step of introducing a first desired DNA into a target cell using Tol1 element and including a further step of introducing a second desired DNA into the target cell using Tol2 element. A DNA introduction method using Tol2 element can be carried as described in Koga A., Hori H., and Sakaizumi M. (2002) Mar Biotechnol 4: 6-11. (Cited document 13), Johnson Hamlet M. R., Yergeau D. A., Kuliyev E., Takeda M., Taira M., Kawakami K., and Mead P. E. (2006) Genesis 44: 438-445. (Cited document 14), Choo B. G., Kondrichin I., Parinov S., Emelyanov A., Go W., Toh W. C., and Korzh V. (2006) BMC Dev Biol 6: 5 (Cited document 15), and the like. In addition, Cited document 13 reports gene introduction using Tol2 element, Cited document 14 reports mutagenesis using Tol2, and Cited document 15 reports trapping of genes and promoters or enhancers using Tol2 element.
The DNA introduction system of the present invention can be used in transgenic fish, transgenic mice, knockout mice, etc., for the purpose of producing genetically modified animals. For example, the DNA introduction system of the present invention can be introduced into a cytoplasm, a vitellus, or a nucleus of a fertilized egg of zebra fish, medaka fish, etc., by a method such as microinjection to generate transgenic fish.
By using the DNA introduction system of the present invention, a specific gene-introduced fertilized oocyte or embryonic stem cell can be produced, and a transgenic non-human mammal can be generated from such a cell. The transgenic non-human mammal can be produced by microinjection in which DNA is directly injected into a pronucleus of a fertilized egg, a method using a retrovirus vector, a method using an ES cell, or the like. A microinjection method will be described below as one example of a production method of a transgenic non-human mammal.
In microinjection, a fertilized egg is first collected from an oviduct of a female mouse whose copulation was confirmed. The fertilized egg is then cultured and the DNA introduction system of the present invention introduces a desired DNA into the pronucleus. After DNA introduction, the egg is transplanted into an oviduct of a pseudopregnant mouse; the mouse is allowed to gestate long enough to obtain a neonatal mouse (P0). To confirm that the desired DNA is correctly incorporated into chromosomes of the neonatal mouse, DNA is extracted from the tail or the like of the neonatal mouse. The extracted DNA is then subjected to PCR using a primer specific to the desired DNA, a dot hybridization method using a probe specific to the desired DNA, or the like. Although a species for “the transgenic non-human mammal” is not particularly limited, rodents such as mice and rats are preferred.

(Method of Transferring Specific DNA Site on Genomic DNA)

Another aspect of the present invention provides a method of transferring a specific DNA to a site on genomic DNA of a target cell by using the transposase of the present invention or a polynucleotide encoding the transposase. In one embodiment of this aspect, the transposase or the polynucleotide is introduced into a cell (target cell) having Tol1 element in which a polynucleotide encoding a transposase includes a deletion. The introduced transposase (or a transposase expressed from the introduced polynucleotide) acts on Tol1 element contained in the target cell to allow transposition. In addition to target cells that obtain Tol1 element artificially, a cell naturally (i.e., as an intrinsic element) having Tol1 element can be used as the “target cell” herein. That is, the method of the present invention is not limited to cells for which an exogenous Tol1 element has been introduced.
For a Tol1 element having insertion of another polynucleotide sequence, the consequences of the other polynucleotide sequence's transfer can be examined, and information about functions of the other polynucleotide sequence can be obtained. Thus, the present invention can be used for analysis of various polynucleotides' functions. For a Tol1 element lacking insertion of another polynucleotide sequence, functions of the Tol1 element itself and influences due to its insertion can be examined. Thus, the method of the present invention is also useful in studies of Tol1 element itself.
A transposase corresponding to either Tol1 element or Tol2 element enables selective transposition of a first desired DNA or a second desired DNA that are introduced into cells, with each desired DNA using one of Tol1 element or Tol2 element. That is, it is possible to control two desired DNAs independently. The present invention thus provides a method of transferring a specific DNA site on genomic DNA, which includes a step of providing a transposase corresponding to Tol1 element or Tol2 element to a target cell. The transposase may be expressed in a target cell in which polynucleotide encoding the transposase has been introduced into the target cell. Regarding introduction of a Tol2 element transposase, Koga A., Hori H., and Sakaizumi M. (2002) Mar Biotechnol 4: 6-11. (Cited document 13), Johnson Hamlet M. R., Yergeau D. A., Kuliyev E., Takeda M., Taira M., Kawakami K., and Mead P. E. (2006) Genesis 44: 438-445. (Cited document 14), Choo B. G., Kondrichin I., Parinov S., Emelyanov A., Go W., Toh W. C., and Korzh V. (2006) BMC Dev Biol 6: 5 (Cited document 15), and the like can be referred to. The amino acid sequence of the Tol2 element transposase is SEQ ID NO: 7. The cDNA sequence (which lacks a poly A) encoding the transposase and the genomic DNA sequence (which lacks a TSD) are SEQ ID NOs: 8 and 9, respectively.

(Genetically Manipulated Cells)

When the DNA introduction system or the DNA introduction method of the present invention is carried out, a genetically manipulated cell is generated. Therefore, the present invention also provides a genetically manipulated cell thus obtained. The cell of the invention can possess new characteristics and functions from the genetic manipulation. Such a cell can be used in production of specific substances, treatments of specific diseases, and the like, depending on the introduced DNA. In addition, the cell can also be used to examine the functions of the introduced DNA.

(DNA Introducing Kit)

The present invention further provides a DNA introducing kit used in the DNA introduction system and the DNA introduction method of the present invention. The DNA introducing kit has a donor plasmid as a transporter of desired DNA and a helper plasmid as a transposase source and source for essential constituent factors. Specifically, the donor plasmid includes an expression construct having a transposase gene-defective Tol1 element and having an insertion site. The helper plasmid includes an expression construct containing the transposase or a polynucleotide encoding the transposase. The “insertion site” herein means a site where a desired DNA is inserted. A restriction enzyme recognition site inherent in Tol1 element can be used as the “insertion site.” For example, Tol1 element depicted in the nucleotide sequence of SEQ ID NO: (Tol1-tyr, 1855 base pairs, not containing TSD) has a SalI recognition site, which can be used as an insertion site. A nucleotide sequence can be insertion into a restriction enzyme recognition site by genetic engineering. The nucleotide sequence for a recombination reaction refers to an attR sequence used in, for example, Gateway™ (Invitrogen Co., Carlsbad, Calif.) technology.
A plurality of restriction enzyme recognition sites may be used as insertion sites. A donor plasmid having a multi-cloning site (MCS) may be used. Although the restriction enzyme recognition sites included in a MCS are not particularly limited, it is preferable to include commonly-used restriction enzyme recognition sites such as HindIII, BamHI, and EcoRI. This is because a kit with high versatility is constructed including these sites. A donor plasmid (pDon253Mcs) shown in examples described below has a MCS including of BamHI, EcoRI, EcoRV, KpnI, PstI, XbaI, etc recognition sites.
As described above, transposition efficiency is not reduced when an internal region of 1592 base pairs (from its 158th base to 1749th base counting from the 5′-end) of Tol1-tyr are removed. Based on this, one embodiment of the present invention uses Tol1 element having a structure in which insertion sites are formed between the 5′-end region DNA and the 3′-end region DNA, obtained by removing at least from its 158th base to the 1749th base counting from the 5′-end in the nucleotide sequence of Tol1-tyr (nucleotide sequence of SEQ ID NO: 10). In other words, the embodiment uses a donor plasmid in which a desired DNA is inserted between DNA in the 5′ end region (157 base pairs at maximum length) of the nucleotide sequence of SEQ ID NO: 10 and DNA in the 3′ end region (106 base pairs at maximum length) of the nucleotide sequence of SEQ ID NO: 10. A specific example of the 5′-end region DNA is DNA having the nucleotide sequence of SEQ ID NO: 21 (157 base pairs), and a specific example of the 3′-end region DNA is DNA having the nucleotide sequence of SEQ ID NO: 22 (106 base pairs).
A preferable embodiment is a kit. The kit includes a first vector, which is used as a donor plasmid, containing Tol1 element in which a polynucleotide encoding a transposase is defective and having an insertion site. The kit also includes a second vector, which is used as a helper plasmid, containing a polynucleotide encoding a transposase. Such a kit has high convenience, and a high DNA introduction efficiency can be expected. The helper plasmid in this case further contains a promoter operably linked to a polynucleotide encoding a transposase and/or a poly-A additional signal sequence or a poly-A sequence connected to the polynucleotide in the downstream side.

(Reconstructed Transposon)

A further aspect of the present invention provides a reconstructed transposon. The reconstructed transposon of the present invention includes a polynucleotide encoding a transposase which is inserted in a transposase gene-defective Tol1 element. Preferably, a promoter operably linked to the polynucleotide encoding a transposase is also inserted in Tol1 element. When the inserted “polynucleotide encoding a transposase” contains a promoter region providing sufficient transcription activity, insertion of another promoter is unnecessary. It is preferable that a poly-A additional signal sequence or a poly-A sequence is also inserted to enhance stability of a transcription product (i.e., mRNA). In one preferable embodiment of the reconstructed transposon of the present invention, a poly-A additional signal sequence or a poly-A sequence is connected to a polynucleotide encoding a transposase in its downstream side.
Insertion of a polynucleotide encoding a transposase, and the like, may be carried out using conventional methods (see Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987)); Molecular Cloning provides definitions for terms used herein, such as “operably linked,” and “promoter,”.
A specific example of a reconstructed transposon is one having a sequence in which the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 is inserted in the nucleotide sequence of any of SEQ ID NOs: 10 to 12.
The reconstructed transposon contains a transposase that can be expressed in a target cell and can function as an autonomous transposable element. Therefore, the reconstructed transposon can be used as a tool for introducing DNA independently. The present invention thus also provides a DNA introduction system using a reconstructed transposon. “Independently” means that it is unnecessary to also use a separately provided transposase; however it does not exclude use of components and elements (such as vector backbones and related reagents) as required for exerting the functions of the reconstructed transposon.
Matters not particularly mentioned in the present specification (such as conditions and operational methods) may be selected following conventional methods, and for example, as taught in Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987).

Example 1

1. Materials and Methods

(1) Fish

Over 30 years ago, a completely albino individual medaka fish was found in a commercially bred group of fish (Cited document 27). A primary line was established from this individual, which was kept in an experimental laboratory. In this albino mutant line, 1.9 kb of Tol1 element is inserted in the first exon of a tyrosinase gene (Cited document 11). In 2001, an individual fish showing mosaic pigmentation appeared in a subline maintained at Niigata University. Pigmentation did not occur in the primary line maintained in Nagoya University. The primary line was called i¹-Tomita and the mosaically-pigmented subline was called i¹-Niigata (Cited document 12). These designations are also referred to as subline A and subline B, respectively. The sublines have not previously been bred with other lines.

(2) Database

The following public databases were used: Genomic project of medaka fish (at the World Wide Web (www) shigen.lab.nig.ac.jp/medaka/genome/): for constructing a nucleotide sequence that is likely the Tol1 element; MOTIF (at the World Wide Web (www) motif.genome.jp/): for searching a motif; Pfam (at the World Wide Web (www) sanger.ac.uk/Software/Pfam/): for collecting sequences of transfer enzymes of the hAT family; and Clustal-X (at the World Wide Web (www) bips.u-strasbg.fr/fr/Documentation/ClustalX/): for checking amino acid sequences.

(3) Reagents and Kits

The following molecular biology reagents and kits were used in accordance with manufactures' instructions: PCR enzyme ExTaq™ (Takara Bio Inc., Otsu, Japan) for PCR amplification of DNA; fosmid vector pCC1FOS™ (EPICENTRE Biotechnologies, Madison, USA) for production of genomic library; AlkPhos Direct™ Labelling and Hybridization System (GE Healthcare, Chalfont St. Giles, UK) for labeling and hybridization analysis of probes; RNeasy® kit (QIA GEN GmbH, Hilden, Germany) for RNA extraction; FirstChoice® RLM-RACE kit (Ambion, Austin, USA) for RACE analysis; PolyFect® Transfection Reagent (QIAGEN GmbH) for incorporation of DNA into cells; and G418 (Invitrogen Corp., Carlsbad, USA) for selection of G418-resistant cells. Experimental conditions are described in the experimental results and description of the drawings sections.

(4) Analysis of Transposition in Mammalian Cultured Cells

Human HeLa cells and mouse NIH/3T3 cells were used. The cells were maintained in an incubator containing 5.0% of CO₂at 37° C. and were cultured in DMEM culture medium containing 10% bovine serum and an antibiotic.
1×10⁵cells were seeded per 35 mm-dish and each culture was incubated for 24 hours. A mixture of plasmid DNAs was adjusted to 1000 ng per dish and allowed to be incorporated into the cells using a PolyFect® reagent. The culture was incubated for an additional 24 hours, the cells were then washed twice with PBS, and fresh culture medium (which lacked plasmid DNA or an incorporation reagent) was added to the culture. After 24 hours, cells were separated from the bottom of the dishes with a trypsin treatment and suspended in 2.0 ml of the culture medium. 100 μl each of the suspension was transferred to dishes of different sizes (35 mm, 60 mm, 90 mm). G418 was added to the culture medium to a final concentration of 500 μg/ml. After 12 days of G418 selection, the cells were fixed in 20% formalin and stained with Giemsa stain. A dish having approximately 100 colonies was selected and the number of colonies was counted. From that number, the colony number per 10⁵cells initially seeded was estimated.
The above-described analyses were carried out by simultaneously preparing three groups of measurement systems.

2. Experimental Results

(1) Breeding of Fish

In the moasically-pigmented subline that was discovered in 2001, the penetrance of pigmentation was 20% (i.e., the ratio of pigmented individuals among all individuals). To produce a subline having higher penetrance, a male and a female fish having strong pigmentation were bred; this was repeated for an additional four generations. As a result, the penetrance of the ultimate subline was 90% or more; in addition, pigmented spots were larger than in the original breeding pair (FIG. 1).
(2) Construction of Sequence Likely to be Full-Length Tol1 Element from Data Analysis
A copy of Tol1 element (Tol1-tyr), which was isolated as an insertion into a tyrosinase gene, is 1.9 kb (SEQ ID NO: 10, containing no TSD) in length. Inverted repeat sequences are present in the 5′-end region and the 3′-end region of Tol1-tyr. The sequence in the inverted repeat sequence in the 5′ end region of a sense strand (direction from 5′-end to 3′-end) is of SEQ ID NO: 5 and the inverted repeat sequence in the 3′ end region of a sense strand (direction from 5′-end to 3′-end) is of SEQ ID NO: 6. A nonautonomous copy is often formed due to deletion of the inside in a DNA type element (Cited document 19). Database searches were conducted to identify a longer copy based on Tol1-tyr. In each search, overlapping sequences were identified; these overlapping sequences, when combined, were considered part of full-length Tol1. Based on the combined overlapping sequences, further searches were performed. The further searches discovered that 4.3 kb was absent from Tol1-tyr. Then, this 4.3 kb sequence was anew checked with the database. For each base position of 4.3 kb, the nucleotide having the highest frequency was included in a consensus sequence. As a result, a 4.3 kb consensus sequence having 2.3 kb of an open reading frame (ORF) was obtained. This sequence is named as Tol1-L0.

(3) Identification of Autonomous Tol1 Element

PCR amplification of an internal 1.2 kb region of the sequence of Tol1-L0 (part of FIG. 2 b) was performed on genomic DNA of a mosaically-pigmented subline (named fish B). A fragment obtained by PCR amplification was used as a probe in colony hybridization of a genomic library from subline B; the colony hybridization identified two clones. The clones were named Tol1-L1 and Tol1-L2. The two clones each included 4.3 kb of sequence and they exhibited an identical restriction enzyme map for five restriction enzymes (data not shown). Since the nucleotide sequences appeared the same, further work was performed using only Tol1-L1. As a result, the nucleotide sequence (SEQ ID NO: 4) and structure of Tol1-L1 were determined (FIG. 2). Information from a further analysis, as described below, and comparison between structures of Tol1-L1 and Tol1-tyr are shown in FIG. 2.
(4) Identification of Full-Length cDNA Likely Encoding Tol1 Transposase
3′ RACE (rapid amplification of cDNA ends) was performed to identify a transcription product of the Tol1 transposase gene. This was done using RNA extracted from an albino line without pigmentation (subline A) and a mosaically-pigmented subline (subline B). By Southern Blot, one signal was observed in subline B and no signal was observed in subline A (FIG. 3). The result indicates that a Tol1 transcription product from an ORF is present in subline B but not in subline A; if an ORF is present in subline A, the amount thereof must be very small. Then, 5′ RACE of subline B was performed and one signal appeared (FIG. 3). Clones of the RACE products were obtained and their nucleotide sequences were examined; from this the cDNA sequence (SEQ ID NO: 3), with a 2.9 kb ORF (SEQ ID NO: 1), was obtained (FIGS. 11 to 13). This sequence was registered in DDBJ/EMBL/GenBank as Accession No. is AB264112. By comparing the sequence of full-length cDNA and the Tol1-L1 sequence (SEQ ID NO: 4), it was revealed that a Tol1 transposase gene is composed of three exons (FIG. 2).
A BLAST search, using the amino acid sequence deduced from Tol1 ORF (SEQ ID NO: 2, FIGS. 11 to 13), revealed a list mainly composed of transposable elements in the hAT family. hAT family elements from rice or Arabidopsis had the highest similarity amongst sequences in the database (data not shown). Further, when the amino acid sequence was reviewed for the presence of common motifs, a dimerization domain (PF05699) of the hAT family was found in the Pfam database. Further, the Tol1 ORF was found to have those amino acids commonly present in Tol1 and hAT elements of other species (FIG. 4). Similarity of the amino acid sequence to Tol2 was lower than similarity to elements included in FIG. 4 (data not shown).

(5) Structure of Medaka Fish Genomic Tol1

The previously-performed database search suggested that, in medaka fish genomes, there is greater variability in the Tol1 internal region when compared to the Tol1 end regions. Southern blot analysis using various medaka fish lines confirmed this. Probes that corresponded to various parts of full-length Tol1-L1 were used. When a probe that hybridized to an end region was used, 100 bands or more appeared; on the other hand, when a probe for an internal region was used, the number of bands was 0 to 5 (FIG. 5). Such a phenomenon is commonly observed also in Activator element of corn (Cited document 19), p element of Drosophila (Cited document 20), and other DNA type elements. It is generally accepted that internal deletion can generate a nonautonomous element from an autonomous element (Cited documents 19 and 20). This may also be applicable to Tol1.

(6) Demonstration of Tol1 Transposition in Mammalian Cells

To determine whether Tol1 ORF encodes a transposase and whether it can mediate a transfer reaction of DNA intervening a Tol1 element, a donor plasmid (hereinafter also referred to as a “donor”) and a helper plasmid (hereinafter also referred to as a “helper”) were prepared. The donor plasmid had 1.9 kb of Tol1-tyr with a neomycin-resistant gene incorporated within Tol1-tyr. The helper plasmid had Tol1 ORF. A CMV promoter was connected upstream to the ORF, and in the same manner, a poly-A additional signal was connected to the downstream side for stabilization (FIG. 6). A defective helper plasmid, which served as a negative control, was prepared. The defective helper plasmid was prepared by replacing two codons of the internal ORF with stop codons. Further, a filler plasmid having an irrelevant DNA fragment but of the same length as Tol1 ORF was prepared. The filler plasmid was used so that the same amount of exogenous DNA was introduced in each experiment. These plasmids were combined and introduced into human HeLa cells or mouse NIH/3T3 cells. Cells that had acquired G418 resistance were selected. A large number of G418-resistant colonies were generated in cells introduced with a donor plasmid and a helper plasmid, compared to cells introduced with a donor plasmid and a defective helper plasmid or a donor plasmid and a filler plasmid (FIG. 7). To confirm that G418 resistance resulted from transposition (incorporation) of Tol1 into a target cell's genome, a DNA fragment containing Tol1 was cloned from a G418-resistant cell introduced with a donor plasmid and a helper plasmid. Then, the nucleotide sequences of a Tol1 end region and a flanking portion were examined. When eight clones were examined, their aligned sequences were significantly different (FIG. 8). Each of the eight clones had two 8 bp target site duplications (TSD). This indicates that incorporation of a Tol1 portion of the donor plasmid into a chromosome results from the transfer reaction. These data demonstrated that Tol1 ORF encodes a functional Tol1 transposase.

(7) Comparison Between Transposition Frequencies of Tol1 and Tol2

Transposition frequencies of Tol1 and Tol2 were examined using HeLa cells. A donor plasmid and a helper plasmid, for each of Tol1 and Tol2, were prepared and co-introduced into the HeLa cells. Ratios of a donor plasmid and a helper plasmid were set within the range of 1:0.5 to 1:9 (when the amount of donor is 100 ng) and within the range of 1:0.5 to 1:4 (when the amount of donor is 200 ng). Within these ranges, transposition frequencies for either element showed positive correlation to the amount of helper plasmid (FIG. 9). The “net colony number” was found by subtracting the “colony number with no helper” from an “observation value of the colony number.” The maximum “net colony number” of Tol1 was 3780−120=3660 (donor 200 ng, helper 800 ng) and that of Tol2 was 3,393−287=3,106 (donor 200 ng, helper 400 ng). The ratio of maximum values (Tol1 maximum value/Tol2 maximum value) was 1.18. Thus, the maximum transposition frequencies of Tol1 and Tol2 were equivalent.

(8) Noninterference Regarding Transposition Induction of Tol1 and Tol2

Both Tol1 and Tol2 are hAT family elements and are present in genomes of the same fish species. Thus, it was examined whether a Tol1 transposase induces Tol2 transposition and whether a Tol2 transposase induces Tol1 transposition. In this experiment, a 1:4 ratio of a donor plasmid and a helper plasmid was introduced into HeLa cells. It was shown in past experiments that this ratio causes high frequency transposition.
The experimental result clearly showed that the Tol1 transposase does not induce Tol2 transposition and that the Tol2 transposase does not induce Tol1 transposition (FIG. 10). Thus, each of the two transposases functions only for its corresponding elements.

3. Discussion

Two types of transposition factors, which are known as transposable elements, are present in genomes of vertebrates; both of which have transposable activities. The transcription factors are Tzf element of zebrafish and Tol2 element of medaka fish. Although transposition of a Tol1 element not directly demonstrated at the time Tol1's discovery, apparently the insertion of a Tol1 element was responsible for mutation of the albino line (subline A) of medaka fish (Cited document 21). Since almost all known copies include internal disruptions or deletions, it was expected that other copies are present in genomes. Here, the present inventors identified an intact Tol1 element from a database search and found that it can cause transposition at a high frequency. Furthermore, the present inventors characterized a subline (subline B) that has a unique mosaic pigmentation.
Tol1 and Tol2 both belong to the hAT family. However, there are large differences in their molecular structure and their distribution among species. Many Tol1s have internal deletion of various sizes; repeat sequences having homology in Tol1 are widely distributed in members of the Oryzias genus (Cited document 10). Unlike Tol1s, Tol2s have uniform sequences and structures and are observed only in medaka fish species (O. latipes) and its related species (O. curvinotus) (Cited document 23). The inventors speculate that Tol1 has been present in the Oryzias genus for a long time; on the other hand, Tol2 recently emerged in the genome of a line related to medaka fish (Cited document 23). It is likely that the two elements coexist in modern-day medaka fish by chance, and a sufficient time have passed; therefore, separate transfer reaction lines are now established.
Mosaic pigmentation observed in Tol1-tyr is similar to the unstable body color mutation that was recently discovered by the present inventors in another gene (Cited document 9). In a line showing unstable body color, it was found that Tol2 excision from a tyrosinase gene occurs at high frequency and Tol2 insertions occur in various sites of a genome. Transposition burst, the bursting of active transposition by a transposable element, is frequently observed in non-vertebrate model organisms. The discovery of transposition burst by Tol2 medaka fish was the first example of transposition burst in vertebrates. Presently, examination of insertion of Tol1 is difficult. This is because there are more copies of Tol1 in medaka fish genomes than copies of Tol2 and it is expected that such examination beyond the capabilities of the currently-used analysis methods. If transposition burst also occurs in Tol1, it is possible that a DNA-type element greatly influenced genomic evolution of vertebrates. Determining the degree of this influence on evolution is required.
Tol1 was believed to have lost its functions until a mosaically-pigmented medaka fish was discovered. This is because, although over 100 Tol1 copies had been examined, none resembling a gene had been found (Cited document 10). From human and other genome sequencing projects, it has been found that a considerable number of DNA transposable elements exists invertebrate genomes. However, most of these elements have lost their transposition activities (Cited document 4). Prior to the present invention, the inventors questioned whether there existed a DNA transposable elements capable of reactivation. Thus, it is significant that the above-described Southern Blot analysis revealed that a potential autonomous copy exists in subline A.
In a comparison experiment of transposition frequencies of Tol1 and Tol2, amounts of introduced donor plasmid and helper plasmid were varied. There, the total weight of plasmid introduced did not vary. However, each plasmid introduced had a different molecular mass; thus, the molar ratios of introduced Tol1 or Tol2 plasmids, that is, the number of molecules, was not the same between conditions. More specifically, the length a transposable element in the donor plasmid and the length of a cDNA in the helper plasmid differed between the two elements. For the donor plasmid, the lengths of the whole element, including the neomycin-resistant gene, were 3.7 kb in Tol1 and 3.1 kb in Tol2. The lengths of the encoding regions in the helper plasmid were 2.8 kb in Tol1 and 2.0 kb in Tol2 (see descriptions of FIGS. 5 and 9). Although there were such differences, comparative transposition efficiencies of Tol1 and Tol2 could be determined from the experimental results. Importantly, the maximum transposition frequency for Tol1 was equal to the maximum transposition frequency for Tol2. Tol2 is an element recently used in genetic modification systems in vertebrates, such as gene introduction (Cited document 13), mutagenesis (Cited document 14), and trapping of genes and promoters or enhancers (Cited document 15). Accordingly, it is likely that Tol1 has similar capabilities. It is extremely important that the two types of elements do not influence each other's transposition. Existence of two DNA introduction systems capable of independent control is particularly beneficial when co-introduction of two desired DNAs into one cell line or organism is necessary. It is also envisaged that a first introduced DNA is transferred by supplying a transposase of an element corresponding to the first DNA and not supply a transpose of an element corresponding to a second DNA.
While both of Tol1 and Tol2 are elements in the hAT family, Sleeping Beauty and Frog Prince, which were derived from fish and frog genomes, belong to the mariner/Tc1 family. piggyBac, which was derived from an insect genome, belongs to another family. A significant difference among these transposable element families lies in the size of an element. Most of the mariner/Tc1 family elements are 1 to 2 kb long, and piggyBac are 2.5 kb long. On the other hand, a typical full-length element in the hAT family is 4 to 6 kb long. Since, in many elements, there is a negative correlation between element length and transposition frequency (Cited document 24), having a large element belonging to the hAT family would be useful for transferring a large DNA fragment. In fact, the present inventors reported that Tol2, which is up to 9.0 kb long, can be transferred (Cited document 13). In addition to an element's length, there is another difference among the transposable element groups, namely, “restriction accompanied by excessive expression.” When a transposase exists in an excessive amount, transposition frequencies decrease in Sleeping Beauty (Cited document 24), mariner of Drosophila (Cited document 25), and piggyBac (Cited document 25). However, such a phenomenon did not appear to occur in Tol1 or Tol2. Others studying Tol2 reported similar results (Cited document 26). It is potentially very useful that two hAT family elements, which independently function and have high transposition frequencies, can be used for genetic manipulation in vertebrates.

Cited Documents

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Example 2

Using an autonomous copy of Tol1 element (Tol-L1, length 4355 bp, DDBJ/EMBL/GenBank Accession No. AB288091, SEQ ID NO: 4) and a transposable enzyme gene (length 2900 bp, DDBJ/EMBL/GenBank Accession No. AB264112, SEQ ID NO: 3) (Example 1), a Tol1 element is now available as a genetic tool for use in mammals.
A common feature of transposable elements is a reduction in transposition frequency as the length of an element decreases. Thus, “loading ability” is important when selecting an element. The “loading ability” herein means the “maximum length of a DNA fragment that can be carried by an element.” Tol1 element is expected to be highly useful in this regard. First, the Tol1 element belongs to the hAT family. The hAT family is a group of transposable elements represented by hobo element of Drosophila, Activator element of corn, and Tam3 element of snapdragon (Cited documents 2 and 16). A particular characteristic of this family is a longer whole (complete) length as compared to other common element families. Specifically, while the hAT family elements have lengths of 4 to 6 kb, most elements in the mariner/Tc1 family are 1 to 2 kb and piggyBAac element are 2.5 kb, which are comparatively short. Second, before the current study, a preliminary examination conducted by the present inventors suggested that Tol1 element exceeding 15 kb exists in medaka fish genomes. Based on these findings, the present inventors inferred that Tol1 element transfers even if the whole length exceeds 15 kb. Tol2 element is also a hAT family element (Cited document 7). However, copies of this element have no structural differences, and almost all copies are 4.7 kb long. Although the present inventors have made large-scale investigations on naturally existing Tol2 element, so far, an element longer than 4.7 kb has not been found (Cited documents 7 and 8).
Here, the inventors investigated the length of naturally-existing Tol1 element. It was found that copies of about 18 kb and about 20 kb in length exist. Consequently, the Inventors set out to develop a gene introduction vector are able to incorporate long DNA fragments into a chromosome. First, an internal region, which is unnecessary for a transfer reaction, was removed from a 1.9 kb element, thereby producing a short vector having a whole length of 0.3 kb. Then, another DNA fragment was inserted into this vector and this second vector served as a basis for preparing Tol1 elements having various lengths. Each Tol1 element was then incorporated into a cell by lipofection. Subsequently, G418 selective culturing was performed and the residual colony number was counted. This number was used to calculate a transposition frequency (see Example 1 for the experimental technique). However, since lipofection may have affected the efficiency of DNA incorporation, a control that excluded the influence of the incorporation efficiency was conducted. The control was to compare efficiency when the sizes of whole plasmids were the same, even when the lengths of Tol1 elements inserted therein differed. This revealed that even when Tol1 element was as long as 22.1 kb, the Tol1 element transferred efficiently. Indeed, this is the longest DNA transposable element reported in mammals.

1. Materials and Methods

(1) Genomic Library

A genomic library of medaka fish was prepared in a previous study (Cited document 10). The library was used to obtain a clone of genomic DNA containing Tol1 element. The original genomic DNA of medaka fish for the library was extracted from an albino medaka fish that had partial melanin pigmentation on its skin and eyes. The vector was fosmid pCC1FOS™ (EPICENTRE Biotechnologies, Madison, Wis., USA), which can accommodate 33 to 48 kb of mechanically-sheared DNA.

(2) Plasmid

Two types of plasmids, i.e. a donor plasmid and a helper plasmid, were used. Tol1 element is excised from the donor plasmid and incorporated into a chromosome due to action of a transfer enzyme encoded by the helper plasmid.
The structure of the donor plasmid used here is shown in FIGS. 15 and 17. The helper plasmid was the same as used in Example 1; its basic structure is shown in FIG. 15. A defective helper plasmid was also prepared. The defective helper acts as a control since it lacks an active transfer enzyme.

(3) Transposition Frequency Measurement System

Human HeLa cells and Mouse NIH/3T3 cells were cultured in a DMEM medium containing 10% FBS (fetal bovine serum). The culture temperature was 37° C. and the CO₂concentration was 5.0%.
2×10⁵cells were seeded per well in a 12-well plate (diameter 22 mm). After 24 hours, 100 ng of the donor plasmid and 900 ng of the helper plasmid were added to each well and were incorporated into the cells using a Lipofectamine™ LTX reagent (Invitrogen Corp., Carlsbad, Calif., USA). After 8 hours, the cells were washed twice with PBS and the medium was replaced with fresh media, which lacked plasmids. After 24 hours, the cells were separated from the dish with trypsin and suspended in 2.0 ml of fresh media. Mediums containing G418 at a concentration of 500 μg/ml was added to dishes of different sizes (35 mm, 60 mm, and 90 mm), and 400 μl of a cell suspension was added thereto. After 12 days of G418 selection, cells were fixed with 20% formalin and stained with Giemsa stain. A dish having about 100 colonies was selected from the dishes and the colony number was counted. The colony number per 10⁵cells initially seeded was determined based on the counted number and dilution factor. The above-described procedure is the steps included in one trial. Such a trial was performed three times for each combination of donor plasmid and helper plasmid.

(4) Technique of Molecular Level Operation

This study is an extension of the study shown in Example 1. Adjustment of genomic DNA, PCR, cloning of PCR products, determination of nucleotide sequences, and colony hybridization were carried out by the same methods and procedures. However, for PCR, LA Taq™ DNA Polymerase (Takara Bio Inc.) was used here, since it efficiently amplifies long stretches of DNA, rather than of Ex Taq® (Takara Bio Inc., Otsu, Japan) as used previously. PCR conditions are described in the corresponding section.

2. Experimental Results

(1) Mutation of Length Shown in Tol1 Copies

100 to 200 copies of Tol1 exist in a medaka fish genome; interestingly, the lengths of the copies are not uniform (Cited document 9). To determine length variations, a genomic library screen was performed with particular attention to the identification of long copies. Two hybridization screens were carried out and a chromosome fragment containing both end regions of Tol1 was recovered. In the first screen, a clone which hybridized to the left end region (1st to 500th bases of SEQ ID NO: 10) of Tol1-tyr was identified. 4×10⁴colonies (this number corresponds to twice the DNA content of a haploid genome) were screened using a probe labeled with alkaline phosphatase. In the primary screen, 161 positive signals were detected. A secondary screen was performed on the 161 colonies identified in the primary screen. Here, the right end region of Tol1-tyr (1356th to 1855th bases of SEQ ID NO: 10) was used as a probe. In the secondary screen, 130 of the 161 colonies had positive signals.
The clones obtained by the screens were subjected to PCR using primers that recognized both end regions of Tol1. This amplified a Tol1 portion contained in each clone. Amplification was observed in 114 out of 130 clones. The distribution of element lengths was approximately mound-shape with a sharp peak at 1 to 2 kb (FIG. 16). It is particularly notable that clones 18 kb and 20 kb long were identified. As shown later, Tol1 element transfers independent of its internal nucleotide sequences, provided that the element has end regions. Therefore, it was expected that the two long Tol1 elements identified would have transferring ability.

(2) Transposable Activity of Short Clones of Tol1

It has been observed that, as long as a transfer enzyme is present, a DNA transposable element retains its transposable activity even though the element lacks a portion of its internal sequence (Cited document 15). The Tol1-tyr element, which is 1.9 kb long, transfers as long as a transfer enzyme is added, although Tol1-tyr lacks a portion of the internal sequence of Tol1-L1 element, which is 4.4 kb long (Cited document 10). Since many elements shorter than 1.9 kb also exist in a medaka fish genome (FIG. 16), it was considered likely that the 1.9 kb long element contains a portion of the element that is unnecessary for transposition. Thus, many shorter clones were prepared and their transposable activities were examined. The method for measuring the number of colonies used in Example 1 was used here. To prepare shorter elements, a PCR primer was used that was orientated outward at an end region of Tol1-tyr. First, an arm of Tol1 and a plasmid having the arm were PCR amplified as one sequential fragment and both ends of the fragment were connected. Then, a neomycin-resistant gene was incorporated into the connected portion (FIG. 15). The resulting donor plasmid was allowed to be incorporated into cultured mouse cells together with a complete or defective helper plasmid (FIG. 15). One clone comprising a left arm with 157 bp and a right arm with 106 bp showed a transposition frequency equal to or greater than that of Tol1-tyr (FIG. 16). Either arm in this clone was further shortened; when an arm was 26 bp long, transposable activity was extremely decreased, and in some cases, transposable activity was lost (FIG. 16).

(3) Preparation of Short Vector Having Cloning Site

A new clone was prepared based on the previous experimental results. The new clone, pDon263Mcs, has 157 bp of the left arm and 106 bp of the right arm of Tol1 element. The new clone, between the arms, had a multiple cloning site (MCS) including the restriction enzyme recognition sites for six frequently-used restriction enzymes (i.e., BamHI, EcoRI, EcoRV, KpnI, PstI, and XbaI) (FIG. 17). The new clone also included a HindIII site outside of the Tol1 element. The HindIII site allows accurate measurement of a transposition frequency, as described below.
(4) Preparation of a Plasmid Having a Constant Whole Length and Having Tol1 Element with Different Sizes in the Inside Thereof.
A first DNA fragment having a length of “x” kb (“x”=0, 5, 10, 15, or 20) and a second DNA fragment having a length of “y” kb (“y”=20−“x”) were prepared using PCR. The first DNA fragment was inserted into an EcoRI site of pDon263McsNeo (inside Tol1) and the second DNA fragment was inserted into the HindIII site (outside Tol1) (FIG. 17). Clones thus prepared were named pDon263McsNeoExHy. In Don263McsNeoExHy, a Tol1 arm was 0.3 kb long, a neomycin-resistant gene was 1.8 kb long, and vector backbone was 2.7 kb long. Accordingly, the distance from the left end to the right end of Tol1 in pDon263McsNeoExHy was (“x”+2.1) kb. The size of the plasmid on the whole was 24.8 kb regardless of the value of “x”.
It is known that a plasmid's size influences its incorporation efficiency by lipofection. Thus, a second DNA fragment was inserted outside Tol1 in addition to first DNA fragment which was inserted inside Tol1. This standardized the size of each plasmid which negated an influence a plasmid's size would have on incorporation efficiency. Thereby, precise comparison of transposition frequencies among donor plasmids with different amounts of Tol1 and/or insert became possible.

(5) Comparison of Transposition Frequencies

A transposition frequency for each of five types of donor plasmids was measured, combining the donor plasmids with a complete helper plasmid or a defective helper plasmid (FIG. 18). Both in human and in mouse cells, the size of an element was negatively correlated with transposition frequency. The ratio of transposition frequency of the longest element (pDon263McsNeoE20) and transposition frequency of the shortest element (pDon263McsNeoH20), when incorporated with a complete helper, was 0.21 in a human cell and 0.28 in a mouse cell. In a human cell, a transposition frequency of a donor plasmid with a complete helper plasmid, in the case of using the longest element, was 8 times higher than a transposition frequency of a donor plasmid with a defective helper; in a mouse cell, it was 10 times higher.

(6) Demonstration of Transposition

Next, a trial was performed to demonstrate that incorporation of Tol1 element into a chromosome was due to a transfer reaction. First, two colonies of mouse cells obtained in the trial with the longest element (pDon263McsNeoE20) were used to establish cell lines. These cell lines (N1 and N2, in which “N” means a neomycin-resistant transformant) were respectively amplified and their genomic DNA was extracted. The extracted DNA was used as a PCR template and an end region of Tol1 and a chromosome region adjacent to the end region were amplified. The amplification was carried out by inverse PCR. A nucleotide sequence of the PCR product was then examined. It was found that 8 bp of a target site duplication was generated in both cell lines (FIG. 19). Generation of target site duplication means that a reaction incorporating a donor DNA into a chromosome was a transfer reaction. A BLAST search of a mouse database, indicated that the nucleotide sequence was incorporated into chromosome 15 and chromosome 5.
Thus, incorporation into chromosomes by transposition was confirmed. However, it was unclear whether the whole Tol1 element, including an internal DNA fragment, was incorporated in chromosomes without generating partial deletions or disruptions. Therefore, a primer recognizing a Tol1 end region and a chromosome region adjacent to the end region was prepared and PCR was performed. The cell line from which genomic DNA was extracted was diploid; thus, insertion of Tol1 likely occurred in only one of the two homologous chromosomes. The incorporated Tol1 element was amplified using a primer designed, as described above. A corresponding site in the other chromosome was never amplified. This is because the primer included sequence recognizing part of the nucleotide sequence of Tol1 element in the 3′-end, a sequence that was absent from the corresponding site in the other chromosome. PCR amplification was only observed for the correct combination of a cell line and a primer (FIG. 20). The length of an amplified product was as expected (i.e., 22.1 kb) and a restriction digest map, obtained by cutting the amplified product with restriction enzymes, was also as expected (FIG. 20). These results demonstrated that the whole region of Tol1 element, 22.1 kb, was incorporated into a chromosome by a transfer reaction without generating deletions or disruptions.

3. Consideration

(1) Variations in the Length of Tol1 Element

In this study, variations in the length of Tol1 element was examined and it was found that copies about 18 kb and about 20 kb long exist in a medaka fish genome. This result indirectly supported the inventors' reasonings that Tol1 element transfers even if the length thereof exceeds 15 kb.
Two genomic library screens and three PCRs on each clone were performed. Other considered methods include (1) analysis of a nucleotide sequence database of medaka fish and (2) PCR of genomic DNA. Although the nucleotide sequence database of medaka fish has continuously improved, at the time of invention, precise incorporation of a long scattered repeat sequence, such as a transposable element, could not be obtained from the database. Continuous sequences such as a contig or scaffold sequence are assembled by a computer, and these sequences are often broken at the inside of long repeat sequences. Actually, autonomous Tol1 element of 4.4 kb, which was previously identified by the present inventors (Cited document 10), has not yet appeared in the database as a train of one sequence (version 46 published in August 2007).
PCR directly using genomic DNA could not be used. This is because the frequency of short copies, e.g., 1 to 2 kb, is far greater than the frequency of long copies in Tol1; importantly, short copies are predominantly amplified in PCR. Accordingly, much cloning and subsequent PCR analyses on each clone was the only feasible means for conducting this study.

(2) Removal of Unnecessary Internal Region

The present inventors constructed a basic Tol1 vector made with 157 bp of the left arm and with 106 bp of the right arm. The vector transferred at a high efficiency and equivalent to that of the original element (1855 bp). Thus, the present inventors succeeded in removing a 1592 bp internal region. This modification produced an increased space for loading a DNA fragment. Further, it is possible that the internal 1592 bp region includes signals that influence the loaded DNA or the host cell; thus, removing this region may obliterate such influence.
It is likely that the arms could be cut shorter. However, such an analysis was not performed since it is unlikely that a significant increase in loading capacity could be obtained for shorter arms, given that when arms are only 26 bp long, the transposition frequency is extremely decreased (FIG. 16) (the increased content becomes (157−26)+(106−26)=211 bp even at maximum). Also, if arms with certain lengths are preserved, the arms can be used in an analysis of an element incorporated by transposition. In many cases, an initial step in such an analysis is cloning an adjacent chromosome region. The main technique thereof is inverse PCR and it is necessary to use a part of the arms as primer regions in the inverse PCR. Further, two or more rounds of nested PCRs may be necessary, each round requiring a different part of the arms as primers targets. Consequently, the present inventors preserved a portion that can be used for a PCR primer. The arm lengths of the basic vector (157 bp and 106 bp) were determined based on such consideration.

(3) Influence of Element Size on Transposition Frequency

Transposition frequencies were measured when a complete helper plasmid and a defective helper plasmid was used. Formation of colonies was observed also when the defective helper plasmid was used. However, as shown in Example 1, when a defective helper plasmid was used, Tol1 element of a colony did not accompany target site duplication; therefore, the colony was generated by random insertion, not by transposition. This explanation is supported by the fact that the colony numbers using defective helper plasmids were approximately the same among five types of donors. Further, this result also indicates that the inventors had negated any influence of a plasmid size on a DNA incorporation efficiency when using lipofection.
It was revealed from an analysis of transformant cells that the whole region of Tol1 element was incorporated into a chromosome by a transposition reaction. The incorporation frequency was significantly high even for the longest Tol1 element (pDon263McsNeoE20), when compared to random incorporation frequency. The length of Tol1 element of this donor plasmid was 22.1 kb of which 0.3 kb was an arm of Tol1. Accordingly, the basic vector (pDon263Mcs) can deliver to a chromosome a DNA fragment with a length up to 21.8 kb. It was also an important finding that the delivered DNA fragment did not have internal deletions or disruptions.
(4) Comparison with Other Transposable Elements
It is known that Sleeping Beauty element loses transposition efficiency when the whole length exceeds 9.1 kb (Cited document 6). It has been found that piggyBac element functions as a gene introduction vector even when the whole length is 14.3 kb (Cited document 3). In the case of Tol2 element, the maximum length reported so far is 10.2 kb (Cited document 1). For the piggyBac element and Tol2 element, it is possible that transposable activities are retained even for lengths longer than previously reported. Currently, 22.1 kb, as now shown by the present inventors with Tol1, is the largest mammalian DNA transposable element. In addition, the basic vector prepared by the present inventors has an arm of Tol1 as short as 0.3 kb. From the above description, it is clear that Tol1 is a useful gene introduction vector that allows long DNAs to be incorporated in mammalian chromosomes.

Cited Documents

1. Balciunas D., Wangensteen K. J., Wilber A., Bell J., Geurts A., Sivasubbu S., Wang X., Hackett P. B., Largaespada D. A., Mclvor R. S., Ekker S. C. (2006) Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet. 2: e169
2. Calvi B. R., Hong T. J., Findley S. D., Gelbart W. M. (1991) Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3. Cell 66:465-471
3. Ding S., Wu X., Li G., Han M., Zhuang Y., Xu T. (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122: 473-483
4. Fraser M. J., Ciszczon T., Elick T., Bauser C. (1996) Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol Biol 5: 141-151
5. Ivics Z., Hackett P. B., Plasterk R. H., Izsvak Z. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91: 501-510
6. Karsi A., Moav B., Hackett P., Liu Z. (2001) Effects of insert size on transposition efficiency of the Sleeping Beauty transposon in mouse cells. Mar Biotechnol 3: 241-245.
7. Koga A., Hori H. (1999) Homogeneity in the structure of the medaka fish transposable element Tol2. Genet Res 73: 7-14
8. Koga A., Iida A., Hori H., Shimada A., Shima A. (2006) Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces. Mol Biol Evol 23: 1414-1419.
9. Koga A., Inagaki H., Bessho Y., Hori H. (1995) Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes. Mol Gen Genet. 249: 400-405
10. Koga A., Shimada A., Kuroki T., Hori H., Kusumi J., Kyono-Hamaguchi Y., Hamaguchi S. (2007) The Tol1 transposable element of the medaka fish moves in human and mouse cells. J Hum Genet. 52: 628-635
11. Koga A., Shimada A., Shima A., Sakaizumi M., Tachida H., Hori H. (2000) Evidence for recent invasion of the medaka fish genome by the Tol2 transposable element. Genetics 55: 273-281
12. Koga A., Suzuki M., Inagaki H., Bessho Y., Hori H. (1996) Transposable element in fish. Nature 383: 30
13. Koga A., Suzuki M., Maruyama Y., Tsutsumi M., Hori H. (1999) Amino acid sequence of a putative transposase protein of the medaka fish transposable element Tol2 deduced from mRNA nucleotide sequences. FEBS Lett 461: 295-298
14. Miskey C., Izsvak Z., Plasterk R. H., Ivics Z. (2003) The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells. Nucleic Acids Res 31: 6873-6881
15. O'Hare K., Rubin G. M. (1983) Structures of P transposable elements and their sites of insertion and excision in the Drosophila melanogaster genome. Cell 34: 25-35
16. Rubin E., Lithwick G., Levy A. A. (2001). Structure and evolution of the hAT transposon superfamily. Genetics 158: 949-957
17. Zagoraiou L., Drabek D., Alexaki S., Guy J. A., Klinakis A. G., Langeveld A., Skavdis G., Mamalaki C., Grosveld F., Savakis C. (2001) In vivo transposition of Minos, a Drosophila mobile element, in mammalian tissues. Proc Natl Acad Sci USA 98: 11474-11478

Example 3

It was shown in Example 2 that Tol1 is able to carry a long DNA fragment in a chromosome and is an excellent genetic tool. Specifically, it was revealed that Tol1 effectively transposes even if the whole length is as long as 22.1 kb and that Tol1 functions as a vector when the total length of its left and right arms is only 263 kb. Further, it has been shown that Tol1 transposes in human cells and in mouse cells in addition to medaka fish (Example 1; Cited document 15). Therefore, is expected that Tol1 has transposable activity in a wide range of vertebrates.
A DNA transposable element transfers via “cut and paste.” “Cut” indicates a process of excising an element from a DNA molecule, such as a chromosome, on which the element is currently carried. “Paste” means incorporation of the excised element into the same or another DNA molecule. Herein, detection of a “cut” is easy because sufficient information can be obtained by a PCR analysis focusing on a specific element. However, demonstration of incorporation, i.e., “paste” is not as easy since the element's incorporation location is unknown beforehand; thus, a marker gene and a complicated detection system are required.
An object of this study is to examine whether excision of Tol1 element occurs in X. laevis. For this object, an indicator plasmid (in which Tol1 element is embedded) and a helper plasmid (for supplying a transfer enzyme in a cell) were prepared. These plasmids were injected into a flog embryo at an early developmental stage and recovered from the embryo after cell division. Subsequently, the indicator plasmid was analyzed by PCR, cloned, and sequenced. The results showed excision of Tol1 element from the plasmid. Various sequences, known as traces, were also observed at a breaking point. The above results revealed that Tol1 element also shows transposase activity in this model animal, which suggested that Tol1 has excellent versatility as a tool for genomic manipulation.
The sequence of the trace was similar to those used in fish and mammals. However, a tendency that a specific nucleotide would appear at a breaking point was observed. Accordingly, it is possible that a DNA repair mechanisms specific to this frog (or to amphibians, in general) exists, and that the above-mentioned tendency reflects the repair mechanisms.

1. Materials and Methods

(1) Plasmids

Two types of plasmids were used: an indicator plasmid and a helper plasmid. The former includes a nonautonomous Tol1 element and the latter includes a transposase enzyme gene controlled by a CMV promoter. In cells, the helper plasmid supplies a tranposase enzyme, which catalyzes transposition of the Tol1 element present in the indicator plasmid.
In addition to a complete helper plasmid, a defective helper plasmid was also prepared to control for functions of the tranposase enzyme. The defective helper plasmid was obtained by replacing two codons (encoding amino acids) in the middle of the tranposase enzyme with stop codons.
Indicator plasmid plnd263GFP contains a 263 kb arm of Tol1 element with and a GFP gene. The complete helper plasmid pHe1851aa encodes a tranposase enzyme of 851 amino acids. The defective helper plasmid pHe1316aa encodes a inactive protein having only 316 amino acids. Their structures are shown in FIG. 21. The GFP gene in plnd263GFP comprises a CMV promoter, a coding sequence of eGFP, and a poly-A additional signal. This GFP gene serves as a marker gene to confirm that DNA injected into an embryo cell is incorporated into a chromosome.

(2) Injection of Frog Embryo and DNA

600 units and 300 units of chorionic gonadotropin (Aska Pharmaceutical, Tokyo, Japan) were injected into a female frog and a male frog, respectively.
The frogs were naturally bred to obtain a fertilized egg. After removing the fertilized egg's jelly layer in 3% cysteine (pH 7.9) and washing the egg with 0.1× Steinberg's solution (Cited document 12), the egg was transferred to 3% FicoII, 0.1× Steinberg's solution. When the fertilized egg had become a four cell embryo, it was injected with 5 nl of plasmid DNA dissolved in a volume of 88 mM NaCl, 15 mM Tris-HCl (pH 8.0) such that the concentration of the indicator plasmid would be 5 μg/ml and the concentration of the helper plasmid would be 50 μg/ml. After DNA injection, the embryo was cultured in 0.1× Steinberg's solution at 20° C. The ratio of the indicator plasmid to the helper plasmid was 1:10; this ratio is similar to that used in Example 1, which showed that the highest transposition frequency in mammalian cultured cells when the ratio was 1:9.

(3) PCR Analysis

DNA was recovered from an embryo having GFP luminescence when the embryos had reached the tailbud stage. To recover DNA, the embryo was placed in and crushed in a digestion solution 100 μl of [10 mM Tris-HCl, 10 mM EDTA (pH 8.0), 200 μg/ml proteinase K], and digested at 50° C. for 12 hours or more. 2 μl of the digestion product was used as a template, and PCR was performed to detect excision. The polymerase used was KOD Plus polymerase (Toyobo, Osaka, Japan). Primers were P1L (208th to 237th bases in GenBank file L09137) and P1R (770th to 741st bases), which correspond to parts of plasmid pUC19. A position where Tol1 element was incorporated was from the 400th to 441st bases, which was interposed between the primers. Concentrations of dNTPs, MgSO₄and the primers were respectively set to 0.2 mM, 2 mM, and 0.5 μM. Conditions of PCR are described in the corresponding section.

(4) Cloning and Sequencing

The PCR product was diluted 1/500 in water, and a second PCR was performed using 1 μl of the diluted PCR product as template. The primers used were P2L (338th to 367th bases of L01937) and P2R (650th to 621st bases). This nested-type PCR facilitates cloning of a first PCR product. The second PCR product was cloned into an EcoRV site of pBluescript II KS(−) (Stratagene, La Jolla, Calif., USA), and was later sequenced using a T3 primer and a T7 primer; for this, an ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA) was used.

2. Experimental Results

(1) Injection of Plasmid into Embryo and Recovery Thereof.
This experiment consists of two sets of A and B. In A, an indicator plasmid (plnd263GFP) and a complete helper plasmid (pHe1851aa) were injected into an embryo of X. laevis from the two-cell to four-cell stages. Set B is a control experiment for determining activity of a transposase enzyme and a defective helper plasmid (pHe1316aa), instead of a complete helper plasmid. In set B, an indicator plasmid was also injected. In set A, DNA was injected into 154 embryos, and in set B, DNA was injected into 168 embryos; 112 embryos and 136 embryos, respectively, survived to the tailbud stage. No obvious difference in survival rates was observed between sets A and B (χ²=3.07, DF=1, P>0.1).
Injected DNA molecules must migrate to a nucleus for excision to occur. This is because transcription occurs in a nucleus and for a transposase gene, which is carried on a helper plasmid, to be transcribed, the transposase must be in the nucleus. To verify this premise, a GFP gene, which is carried on a indicator plasmid was used. When GFP is expressed, it means that a the indicator plasmid had migrated into a nucleus. 57% (64/112) of the embryos of set A expressed GFP and 65% (88/136) of the embryos of set B expressed GFP. There was no apparent difference between these frequencies (χ²=1.48, DF=1, P>0.4). There was also no obvious difference in special patterns of GFP expression when the embryos were observed under a microscope. 12 embryos having stronger GFP expression (A1 to A12) were selected from the embryos of set A; 12 embryos (B1 to B12) were also selected from the embryos of set B. Plasmid DNA was individually recovered from these 24 embryos.

(2) PCR Analysis of Recovered Plasmids

Two PCR protocols were performed on the recovered DNA. The primers used were P1L and P1R (placed in a position where Tol1 element was interposed between plnd263GFP).
The first PCR protocol was to confirm that a indicator plasmid was recovered. The distance between the two primers on plnd263GFP was 2.4 kb, and a product having this length was confirmed in all samples (FIG. 22, upper column). Even though there were different amounts of the products among samples, there was no clear difference in product amounts, overall, between set A and set B. These results confirmed that the indicator plasmid was recovered. If Tol1 element excision from plnd263GFP occurred precisely, a 535 bp PCR product should have been amplified; however, no product of this size was found in any sample.
The second PCR protocol had a shortened elongation step (40 seconds rather than 150 seconds as used in the first PCR protocol) (FIG. 22, lower column). A 40 second elongation step is not sufficient for amplifying the whole region of Tol1 element, and thus, it was expected that a product resulting from excision could be PCR amplified. A PCR product having a size close to 535 bp was observed in all samples of set A (A1 to A12). In contrast, in no sample from set B, was such a PCR product observed. Thus, dropout of Tol1 element from plnd263GFP occurred in embryos of set A but not in embryos of B.

(3) Analysis of PCR Products' Nucleotide Sequences

Nucleotide sequences of the PCR products obtained from 12 embryos of set A were analyzed to examine positions and shapes of break points generated in plnd263GFP. For this analysis, PCR products were again amplified by a nested-type PCR. The primers used were P2L and P2R. Subsequently, PCR products were cloned into plasmids. At this time, only one colony (clone) among the generated colonies was randomly selected for each embryo. Accordingly, the 12 samples in these experiments were all generated by separate excisions. FIG. 23 shows the aligned nucleotide sequences obtained from the 12 samples. In the 12 samples, all or most of the Tol1 element sequence disappeared. Thus, in frog cells, excision of Tol1 element occurred simultaneous with an indicator. Whole regions of Tol1 element were excised in 11 of the 12 samples (A1 to A6 and A8 to A12) and with only 1 to 7 nucleotides corresponding to a part of TSD remaining. 39 nucleotides in the right end of Tol1 element remained in sample A7, and 77 nucleotides in a chromosome region adjacent to the left side of Tol1 disappeared. A part corresponding to one TSD was included in these 77 nucleotides. It was further found that new guanosine (G) residues were introduced in 7 out of 12 samples, either by insertion of a G or by replacement of another nucleotide with a G.

3. Discussion

In this study, an indicator plasmid (plnd256GFP) was injected into an embryo from the two-cell to four-cell stages along with a complete helper plasmid (pHe1851aa), for set A, or a defective helper plasmid (pHe1316aa), for set B. There was no apparent difference in expression frequencies and distribution patterns of GFP in tailbud stages between set A and set B. Therefore, there does not appear to be a clear difference in incorporation efficiencies of DNA into nuclei between set A and set B. Additionally, amounts of indicators recovered from embryos were equal between set A and set B. However, there was a clear difference in PCR products between samples from set A and set B, indicating excision occurred in set A but not in set B. Accordingly, this clear difference is due to a difference in the nucleotide sequences between the complete helper plasmid and the defective helper plasmid. This difference being only an internal 6-nucleotide region corresponding to codons for the 317th and 318th amino acids in pHe1851aa, which are replaced with two stop codons in pHe1316aa. The above results indicate that dropout of the Tol1 portion from the indicator is due to activity of an enzyme encoded by pHe1851aa, whereas a protein encoded by pHe1316aa does not have such activity.
It can be concluded from the above results that Tol1 element causes excision in X. laevis cells. This conclusion is supported by evidence that various traces (i.e., addition or omission of nucleotides of the transferred fragment) accompanied dropout of Tol1 fu. This is because such traces have been observed in many other DNA transposable elements, with examples including hobo of Drosophila (Cited document 1), Activator of corn (Cited document 17), Tam3 of snapdragon (Cited document 4), mariner of Drosophila (Cited document 2), and Tc1 of nematode (Cited document 13).
It is interesting that G residues (cytidine (C) residues in complementary strands) were introduced in 7 samples. Since an analysis of nucleotide sequences was carried out in both strands, it is unlikely that this phenomenon is an artifact due to an experimental method. Since the phenomenon occurred in separate excisions of no less than 7 samples, it is possible that DNA repair mechanisms specific to this specific frog (or to amphibians, in general) exists and that the phenomenon reflect these mechanisms. Although the present inventors analyzed PCR products in 20 or more medaka fish and 20 or more mammalian cultured cells so far, this has not before been observed.
Excision is only a part of a transfer reaction of a DNA transposable element. However, hAT family elements transfer in an unreplication method, that is, a method of inserting an excised fragment into another site (Cited document 10). Understanding the transfer reaction, on the whole, in X. laevis cells will surely be realized.
Among the 12 excisions examined, there was no sample in which a nucleotide sequence accurately returned to its original state. However, this does not signify that the dropped elements are inaccurately incorporated into chromosomes. It is a phenomenon frequently observed in a DNA transposable element that, although the element is precisely cut out at an end and incorporated into a new place, there is an addition or omission of nucleotides upon transfer into a chromosome. The causes may be due to double-strand break repair (Cited document 13), nonhomologous recombination (Cited document 16), and the like. The present inventors recently cloned two Tol1 elements newly inserted in mouse chromosomes. In this example, the first to last nucleotides of the element were exactly cloned with 8 bp of TSD.
DNA transposable elements such as Sleeping Beauty and Tol2 have been used as tools for genomic manipulation in frogs (Cited documents 14 and 5). However, Tol1 has a characteristic which is superior to those elements, i.e., Tol1 can carry a long DNA fragment (see Example 2). Accordingly, Tol1 is not merely an additional genetic manipulation technique useful for frogs, but should be recognized as a useful tool for further experimentation and insights.

Cited Documents

1. Atkinson, P. W., Warren, W. D. & O'Brochta, D. A. (1993). The hobo transposable element of Drosophila can be cross-mobilized in houseflies and excises like the Ac element of maize. Proceedings of the National Academy of Sciences of the USA 90, 9693-9697.
2. Bryan, G., Garza, D., Hartl, D. L. (1990). Insertion and excision of the transposable element mariner in Drosophila. Genetics 125, 103-114.
3. Cary, L. C., Goebel, M., Corsaro, B. G., Wang, H. G., Rosen, E., & Fraser, M. J. (1989). Transposon mutagenesis of baculoviruses: analysis of Trichoplusiani transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virologyl 72, 156-169.
4. Coen, E. S., Carpenter, R. & Martin, C. (1986). Transposable elements generate novel spatial patterns of gene expression in Antirrhinum majus. Cell 47, 285-296.
5. Hamlet, M. R., Yergeau, D. A., Kuliyev, E., Takeda, M., Taira, M., Kawakami, K. & Mead, P. E. (2006). Tol2 transposon-mediated transgenesis in Xenopus tropicalis. Genesis 44, 438-445.
6. Ivics, Z., Hackett, P. B., Plasterk, R. H. & Izsvak, Z. (1997). Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501-510.
7. Koga, A., Shimada, A., Kuroki, T., Hori, H., Kusumi, J., Kyono-Hamaguchi, Y. & Hamaguchi, S. (2007). The Tol1 transposable element of the medaka fish moves in human and mouse cells. Journal of Human Genetics 52, 628-635.
8. Koga, A., Suzuki, M., Inagaki, H., Bessho, Y. & Hori, H. (1996). Transposable element in fish. Nature 383, 30.
9. Koga, A., Inagaki, H., Bessho, Y. & Hori, H. (1995). Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes. Molecular and General Genetics 249, 400-405.
10. Kunze, R. (1996). The maize transposable element Activator (Ac). In: H. Saedler and A. Gierl (ed.) Transposable Elements. Springer, Berlin. pp. 161-194.
11. Miskey, C., Izsvak, Z., Plasterk, R. H. & Ivics, Z. (2003). The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells. Nucleic Acids Research 31, 6873-6881.
12. Peng, H. B. (1991). Appendix A. Solutions and protocols. In: Kay, B. K. and Peng, H. B. (eds) Xenopus laevis: Practical Uses in Cell and Molecular biology. Academic Press, San Diego, pp. 661-662.
13. Plasterk, R. H. (1991). The origin of footprints of the Tc1 transposon of Caenorhabditis elegans. EMBO Journal 10, 1919-1925.
14. Sinzelle, L., Vallin, J., Coen, L., Chesneau, A., Du Pasquier, D., Pollet, N., Demeneix, B. & Mazabraud, A. (2006). Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system. Transgenic Research 15, 751-760.
15. Tsutsumi, M., Imai, S., Kyono-Hamaguchi, Y., Hamaguchi, S., Koga, A. & Hori, H. (2006). Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol-1 transposable element from the tyrosinase gene. Pigment Cell Research 19, 243-247.
16. Weinert, B. T., Min, B. & Rio, D. C. (2005). P element excision and repair by non-homologous end joining occurs in both G1 and G2 of the cell cycle. DNA Repair 4, 171-181.
17. Wessler, S. R., Baran, G., Varagona, M. & Dellaporta, S. L. (1986). Excision of Ds produces waxy proteins with a range of enzymatic activities. EMBO Journal 5, 2427-2432.
18. Wu, S. C., Meir, Y. J., Coates, C. J., Handler, A. M., Pelczar, P., Moisyadi, S. & Kaminski, J. M. (2006). piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and Mosl in mammalian cells. Proceedings of the National Academy of Sciences of the USA 103, 15008-15013.

Example 4

A transfer reaction is one kind of DNA nonhomologous recombination which requires the combined actions of endonuclease, polymerase, ligase, etc. It is still unclear whether a single transfer enzyme performs each of these activities. That is, it is possible that the transfer enzyme partly relies on a host cell for certain required activities. A significant question, from both an evolutionary and biotechnological viewpoints, is whether a required factor is inherent to a host species or is present in a wide range of organisms. From an evolutionary viewpoint, this allows speculation regarding the frequency of transfer of transposable elements among species, also known as “horizontal transfer”. Frequent horizontal transfer is known to be a significant contributor to evolution. From a biotechnological viewpoint, it is important to determine whether a new gene introduction system, etc., is applicable to a wide range of species. In any case, the fewer required host factors inherent to biological species, the wider range of species for which the new gene introduction system can be used. It is speculated from the above-described results (Examples 1 to 3) that Tol1 element has a transposable activity in vertebrates, in general.
As shown in FIG. 24, higher animals diverged into two large phyletic lines in an early stage of evolution. The two phyletic lines are protostomes and deuterostomes. In protostomes, a blastopore, which is generated during an embryo's early development, ultimately becomes the mouth; in deuterostomes the blastopore ultimate becomes the anus. Vertebrates are deuterostomes. Experiments were undertaken to determine whether Tol1 element transfers in protostomes; for this, the inventors used the silkworm, an insect.

1. Materials and Methods

(1) Overview of Methods

The overall procedure is shown in FIG. 25. DNA and RNA used and details of each step are described below.

(2) Transfer Enzyme RNA

Two plasmids (pTem851aa and pTem316aa), shown in FIG. 26, were constructed. RNAs (mRNA851aa and mRNA316aa) were synthesized using RiboMAX™ Large Scale RNA Production System (Promega Corp., Madison, Wis., USA) from pTem851aa and pTem316aa, respectively. mRNA851aa encodes the entire Tol1 transfer enzyme, i.e., 851 amino acids. mRNA316aa encodes up to the Tol1 transfer enzyme's 316th amino acid. mRNA316aa serves as a negative control. These two mRNAs have the same length. Their nucleotide sequences differ only in 6 bases in their middles, with pTem316aa having two stop codons rather than codons for Met and Lys.

(3) Donor Plasmids

A donor plasmid as shown in FIG. 27 was constructed. This was obtained by cloning a part of a tyrosinase gene of an albino medaka fish. It contains 1855 bp of Tol1 element. It was expected that a transfer enzyme encoded by the native silkworm genome can cut out Tol1 element, and that the Tol1 element then transfers into a silkworm cell's chromosome. Here, a transfer enzyme gene is not included in the cloned 1855 bp (Cited document 5). Bacteria containing the donor plasmid were cultured in a liquid medium and plasmid DNA was extracted and purified using the QIAGEN® Plasmid Maxi Kit (QIAGEN GmbH, Hilden, Germany).

(4) Setting of Treatment Section

Three treatment conditions (A, B and C) were performed. In condition A, mRNA851aa and the donor plasmid were together injected into a fertilized silkworm egg; here, transposition should occur. In condition B, mRNA316aa and the donor plasmid were together injected into a fertilized silkworm egg; here, transposition should not occur since a transfer enzyme is incomplete. Condition C is a negative control and DNA or RNA was not injected into a fertilized egg.
(5) Injection into Silkworm
In conditions A and B, a donor plasmid and RNA were mixed so as to have final concentrations of 40 ng/μl and 160 ng/μl, and injected into fertilized eggs using a glass needle. Injection occurred within 40 minutes of the fertilized eggs being laid. Injection was performed on 250 fertilized eggs in condition A and 50 fertilized eggs in condition B. After injection, the fertilized eggs were stored in a plastic box. In condition C, 50 fertilized eggs (which were not injected with DNA and RNA) were stored in the same plastic box. The plastic box was kept warm, at 25° C., to promote development.

(6) Recovery of Plasmid DNA

After 5 to 6 hours of warming, 75, 25, and 25 embryos, respectively, were collected from conditions A, B and C. Groups of 25 embryos were placed into separate centrifuge tubes. The 75 embryos of condition A were divided into three sets of 25, which were named A1, A2, and A3. The remaining embryos of condition A continued to be kept warm at 25° C. Using the Hirt method (Cited document 3), DNA was extracted from embryos of each group. Cyclic DNA, e.g., plasmids, can be efficiently extracted by this method.

(7) Detection of Excision

PCR revealed whether Tol1 element was excised from recovered donor plasmids. The distance between primers Pex1 and Pex2 on the donor plasmid was 2.2 kb. If excision of Tol1 element occurred while the donor plasmid was present in a silkworm cell, the distance between Pex1 and Pex2 would be shortened. Therefore, a PCR product shorter than 2.2 kb indicates that excision had occurred. If only the Tol1 element part was precisely drawn out, the size of the PCR product would be 0.3 kb since Tol1 element is 1.9 kb long.

(8) Extraction of Genomic DNA

After 96 to 97 hours of warming at 25° C., 100 embryos of condition A were collected. Then, genomic DNA was extracted using standard methods: digestion with SDS and Proteinase K and DNA purification with salt and ethanol precipitation (Cited document 7). The obtained DNA was used for detection of insertion. The above-mentioned DNA extraction was performed after detection of excision because it was expected that the copy number a donor plasmid decrease over time as an embryo's cells duplicate. The donor plasmid has a region corresponding to a PCR primer used in insertion detection. Therefore, a PCR product independent from the insert would be generated. Accordingly, as the copy number of a donor plasmid decreases sensitivity of insertion detection is expected to improve.

(9) Detection of Insertion

Detection of insertion was performed using inverse PCR. Tol1 element contained in the donor plasmid lacks an EcoRI recognition site. If a Tol1 element is transferred into a silkworm chromosome and its genomic DNA is cut with EcoRI, then a DNA fragment including the Tol1 element would be cut out from the chromosome. T4 DNA ligase connects both ends of the EcoRI-cut DNA fragment to produce a circular DNA molecule. Primers Pin1 and Pint are annealed to both ends of Tol1 element and directed outward. PCR performed using these primers on the circular DNA molecules generates a PCR product corresponding to the length of the DNA inserted into the silkworm chromosome. The above-described sequential operations were performed to examine whether Tol1 element was inserted into a chromosome.

(10) Cloning and Sequencing

PCR products obtained in excision detection and insertion detection were cloned into a plasmid and the nucleotide sequences were mapped. The plasmid used for cloning was pT7Blue-2 (Takara Bio Inc., Otsu, Japan). A single-stranded DNA primer which annealed to a location about 100 bp upstream to the cloning point was used for sequencing.

(11) PCR Conditions

PCR was used in the above-described analysis. Ex Taq™ (Takara Bio Inc.) was used as the DNA polymerase. PCR conditions are described below.

2. Results

(1) Detection of Excision

For PCR templates, DNA was used which was extracted from a silkworm embryos that were warmed for 5 to 6 hours. PCR primers (Pex1 and Pex2), which are located so as to interpose Tol1 element therebetween on a donor plasmid, were used. PCR products were electrophoresed as shown in FIG. 28. PCR elongation was either for 150 seconds or 20 seconds. 150 seconds is sufficiently long to amplify 2.2 kb, which would include the whole region of Tol1 element on the donor plasmid. 2.2 kb PCR products were amplified from DNA extracted from condition A or B embryos, but not from embryos of condition C. Thus, 2.2 kb PCR products were derived from the injected donor plasmid, not from genomic DNA of a silkworm. The results further showed that the donor plasmids were recovered from embryos of either condition A or B.
Elongation for 20 seconds was carried out to efficiently amplify a PCR product from a DNA molecule in which excision had occurred. PCR products from DNA of embryos of set A1, A2, or A3 were around 0.3 kb long. 0.3 kb PCR products were not observed from DNA of embryos of condition B or C. These results suggested that excision of Tol1 element occurred only in the embryos of condition A.

(2) Confirmation of Excision

PCR products from A1, A2 and A3 were purified by ethanol precipitation and then ligated into plasmid vectors to form clones. One clone was randomly selected from each set and its nucleotide sequence was examined. Their sequences are aligned in FIG. 29. As seen in the Figure, Tol1 element regions were absent in all three clones. Further, a part of each side's target site duplication (TSD) region remained. Then, 8 to 80 bp of newly added DNA was located therebetween. Nucleotide sequences of newly added DNAs were compared to the whole nucleotide sequence of Tol1 element, but no homology portion was identified. The nucleotide sequence of TSD is CCTTTAGC, and its complementary sequence is GCTAAAGG. In many of the newly added DNAs, whole or apart of this complementary sequence seems to be continuous.
The cloning and sequencing of a PCR product clearly indicates that the whole region of Tol1 element disappeared. Thus confirming that excision of Tol1 element from the donor plasmid had occurred.

(3) Detection and Confirmation of Insertion

Genomic DNA was extracted from an embryo of condition A which was warmed for 96 to 97 hours. The genomic DNA was EcoRI digested and circularized as described above. Then, inverse PCR was performed and the product was cloned into a plasmid. When several tens of clones were obtained as bacterial colonies, two colonies were randomly selected. Plasmid DNA was extracted and sequenced. The corresponding portions are aligned in FIG. 30.
In three samples, nucleotide sequences of Tol1 element portions were confirmed and portions outside Tol1 element were not identical. When only the sequences of the outside portions were taken out from clone of a silkworm and checked with the nucleotide sequence database of silkworms (KAIOKOBLAST; See the World Wide Web (www) kaikoblast.dna.affrc.go.jp/), it was found that sequences having 90% or more homology was present in the silkworm genome. In addition, TSD was not found.
From analysis of a nucleotide sequence of an inverse PCR product, a Tol1 element portion was shown to be connected to a silkworm chromosome. Thus, insertion of Tol1 element occurred in a silkworm.

3. Discussion

At an early stage of evolution, higher animals diverged into two large phyletic lines: protostomes and deuterostomes. Vertebrates, such as humans and medaka fish, are deuterostomes. Tol1 element is a DNA transposable element present in a genome of medaka fish, and was speculated to have a transposable activity in other vertebrates. Using silkworms as a model, this study examined whether the Tol1 element transfers in protostomes. The study clearly showed that transposition occurred in an exemplary protostome.
An enzyme catalyzing transposition of Tol1 element is a transfer enzyme of Tol1 element. However, it was uncertain whether a transfer reaction requires only this enzyme or whether other factors, present in a host cell, are necessary. The above-described results that Tol1 element transfers in a protostome inspired further analyses as to whether other factors are present in a host cell. In these further analyses, it was discovered that other factors present in a host cell are not necessary in a transfer reaction of Tol1 element; alternatively, even if other factors are necessary, they are present in cells of both protostomes and deuterostomes.
The above results have a significant meaning also in the field of biotechnology. First, the fact that Tol1 element transfers in a silkworm means that the methods of gene introduction, gene trapping, and mutagenesis, which are used in a silkworm, can be performed using Tol1 element. Further, as described below, it is likely that methods using Tol1 element have properties that exceed those of methods already developed.
In a silkworm, a method using piggyBac element (Cited document 9) and a system using Minos element (Cited document 11) have already been developed. Both of these elements belong to the mariner/Tc1 family. The mariner/Tc1 family is a group of transposable elements, which are similar in their structures and transfer mechanisms, that are present in a wide distribution of organisms. The group was named for the mariner element of Drosophila and the Tc1 element of nematode. In addition to the mariner/Tc1 family, another large group of transposable elements is called the hAT family, which includes hobo element of Drosophila, Activator element of corn, and Tam3 element of snapdragon (Cited document 1). Tol1 element of medaka fish, which was shown above to transfer in a silkworm, belongs to the hAT family (Cited document 6). The hAT family elements transfer even when the whole length is long, as compared to elements of the mariner/Tc1 family. For example, in an experiment using a cultured mouse cell, it was shown that when Sleeping Beauty element (a mariner/Tc1 family element) has a whole length exceeding 9.1 kb, transposable activity is nearly lost (Cited document 12). On the other hand, Tol1 element transfers in a cultured mouse cell at a high frequency even when the Tol1 element has a whole length of 22.1 kb (Cited document 4). Consequently, its use as a vector for introduction of a long DNA fragment into a chromosome is strongly expected. Industrially-useful genes with large whole lengths, such as a fibroin gene, are present in a silkworm and its related species (Cited document 8). For such genes, Tol1 element is expected to be useful vector.
The significance of the above-described results is not limited to the field of biotechnology in regards to silkworms. This is because, it is unlikely that silkworms, among the protostomes, are unique regarding transposition of Tol1 element. Thus, it is expected that Tol1 element will transfer also in other protostomes. Accordingly, it is expected that methods such as gene introduction, gene trapping, and mutagenesis, using Tol1 element, can be applied to a wide range of organisms.

Cited Documents

1. Calvi B. R., Hong T. J., Findley S. D., Gelbart W. M. (1991). Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3. Cell 66: 465-471.
2. Hikosaka A., Koga A. (2007). PCR detection of excision suggests mobility of the medaka fish Tol1 transposable element in the frog Xenopus laevis. Genet. Res.: in press.
3. Hirt B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26: 365-369.
4. Koga A., Higashide I., Hori H., Wakamatsu Y., Kyono-Hamaguchi Y., Hamaguchi S. (2007b). The Tol1 element of medaka fish is transposed with only terminal regions and can deliver large DNA fragments into the chromosomes. J. Hum. Genet. 52: 1026-1030.
5. Koga A., Inagaki H., Bessho Y., Hori H. (1995). Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes. Mol. Gen. Genet. 249: 400-405.
6. Koga A., Shimada A., Kuroki T., Hori H., Kusumi J., Kyono-Hamaguchi Y., Hamaguchi S. (2007a). The Tol1 transposable element of the medaka fish moves in human and mouse cells. J. Hum. Genet. 52: 628-635.
7. Sambrook J., Russell D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 8. Sezutsu H., Yukuhiro K. (2000). Dynamic rearrangement within the Antheraea pernyi silk fibroin gene is associated with four types of repetitive units. J. Mol. Evol. 51: 329-338.
9. Tamura T., Thibert C., Royer C., Kanda T., Abraham E., Kamba M., Komoto N., Thomas J. L., Mauchamp B., Chavancy G., Shirk P., Fraser M., Prudhomme J. C., Couble P. (2000).
10. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat. Biotechnol. 18: 81-84.
11. Uchino K., Imamura M., Shimizu K., Kanda T., Tamura T. (2007). Germ line transformation of the silkworm, Bombyx mori, using the transposable element Minos. Mol. Genet. Genomics 277: 213-220.
12. Karsi A., Moav B., Hackett P., Liu Z. (2001) Effects of insert size on transposition efficiency of the Sleeping Beauty transposon in mouse cells. Mar Biotechnol 3: 241-245.

INDUSTRIAL APPLICABILITY

The present invention provides a Tol1 element transposase, a DNA introduction system using the same, and the like. The present invention is intended for applications such as gene introduction, gene targeting, mutagenesis, trapping of genes, promoters, enhancers, etc.
The present invention is not limited to the description of the above embodiments and examples of the invention. Various modified forms within the range where a skilled person can easily conceive of are also included in the invention without departing from the description of the scope of claims for the patent.
Entire contents of treatises, unexamined patent publications, patent publications, and the like, indicated in the present specification are hereby incorporated by reference.

Claims

1-34. (canceled)

35. A Tol1 element transposase comprising any of proteins selected from the group consisting of:

(a) a protein having an amino acid sequence encoded by the base sequence of SEQ ID NO: 1; and

(b) a protein having the amino acid sequence of SEQ ID NO: 2.

36. A DNA introduction system comprising:

(a) a donor factor having such a structure that desired DNA is inserted in a transposase gene-defected Tol1 element; and

(b) a helper factor containing the transposase according to claim 35.

37. The DNA introduction system according to claim 36, wherein the Tol1 element has the inverted repeat sequence of SEQ ID NO: 5 in the 5′ end region and the inverted repeat sequence of SEQ ID NO: 6 in the 3′ end region.

38. The DNA introduction system according to claim 36, wherein the Tol1 element comprises DNA having the base sequence of any of SEQ ID NOs: 10 to 12.

39. The DNA introduction system according to claim 36, wherein the Tol1 element comprises 5′ end side DNA and 3′ end side DNA obtained by deleting at least from the 158th base to the 1749th base counting from the 5′ end in the base sequence of SEQ ID NO: 10.

40. The DNA introduction system according to claim 36, wherein the Tol1 element comprises DNA having the base sequence of SEQ ID NO: 21 and DNA having the base sequence of SEQ ID NO: 22.

41. The DNA introduction system according to claim 38, wherein a target site duplicated sequence is connected to the 5′ end and the 3′ end of the toll element.

42. The DNA introduction system according to claim 41, wherein the target site duplicated sequence comprises the sequence of any of SEQ ID NOs: 13 to 15.

43. The DNA introduction system according to claim 36, wherein the desired DNA is a gene.

44. The DNA introduction system according to claim 36, wherein the donor factor is a vector obtained by inserting a desired DNA in a transposase gene-defected Tol1 element, and the helper factor is a vector comprising a polynucleotide encoding a Tol1 element transposase, the polynucleotide comprising any of the nucleotide sequences selected from the group consisting of:

(a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; and

(b) the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4.

45. The DNA introduction system according to claim 44, wherein the vector being the helper factor further comprises a promoter operably linked to the polynucleotide.

46. The DNA introduction system according to claim 44, wherein the vector being the helper factor further comprises a poly-A sequence connected to downstream of the polynucleotide.

47. A DNA introduction method comprising a step of introducing the DNA introduction system according to claim 36 to a target cell which is a vertebrate cell.

48. The DNA introduction method according to claim 47, wherein the target cell is a vertebrate cell other than a cell in a state of a constituent factor of a human individual.

49. The DNA introduction method according to claim 47, further comprising a step of introducing DNA different from the desired DNA introduced by the DNA introduction system to the target cell by utilizing Tol2 element.

50. A method of transferring a specific DNA site on genomic DNA, comprising a step of supplying a transposase corresponding to Tol1 element or Tol2 element to a cell genetically manipulated with the DNA introduction method according to claim 49.

51. A method of transferring a specific DNA site on genomic DNA, comprising a step of introducing the transposase according to claim 35 into a cell having a transposase gene-defected Tol1 element on genomic DNA.

52. The method according to claim 51, wherein another polynucleotide sequence is inserted in the Tol1 element.

53. A cell genetically manipulated by the DNA introduction system according to claim 36.

54. A DNA introducing kit, comprising:

a donor factor comprising an expression construct containing a transposase gene-defected Tol1 element and having an insertion site; and

a helper factor comprising an expression construct containing the transposase according to claim 35.

55. The DNA introducing kit according to claim 54, wherein the toll element comprises a structure having the insertion site between 5′ end side DNA and 3′ end side DNA, which is obtained by deleting at least from the 158th base to the 1749th base counting from the 5′ end in the base sequence of SEQ ID NO: 10.

56. The DNA introducing kit according to claim 54, wherein the Tol1 element comprises a structure having the insertion site between DNA having the base sequence of SEQ ID NO: 21 and DNA having the base sequence of SEQ ID NO: 22.

57. The DNA introducing kit according to claim 54, wherein the insertion site is made of a plurality of different kinds of restriction enzyme recognition sites.