JP2007259775A - Efficient method for producing transgenic silkworm - Google Patents

Abstract

A method for producing a transgenic silkworm with high efficiency is provided.
The transposon into which an arbitrary gene is inserted and mRNA of the transferase are injected into a silkworm egg, and a silkworm is produced from the silkworm egg, thereby significantly increasing the production efficiency of the transgenic silkworm. That is, the production efficiency of transgenic silkworms is remarkably increased by using in vitro synthesized mRNA instead of the conventionally used helper plasmid as a source of transposon piggyBac transferase.
[Selection figure] None

Description

  The present invention relates to a method for producing a recombinant silkworm having high efficiency.

  Methods for producing useful substances using genetically modified organisms are widely used in Escherichia coli, yeast, plants, mammals and the like. In recent years, a silkworm, a kind of insect, has developed a technique for producing a recombinant silkworm using a DNA-type transposon, and it has become possible to produce a recombinant protein of a heterologous organism using the silkworm. Silkworms are the only domesticated insects. That is, it can survive only by being kept by humans, and it will die in a few days in nature. In addition, the larvae stay at the place where food is present, and the moth has no power to fly, so the probability of spreading out of the breeding place is very low. Silkworms also have a special tissue called the silk gland that has been differentiated to produce silk, and a large amount of protein can be produced by using this tissue. Such a characteristic of silkworm is advantageous as a recombinant organism for producing useful substances, and it is predicted that many recombinant proteins will be produced using silkworm in the future and used for test drugs and pharmaceuticals. The

  However, the recombinant silkworm production method (Tamura et al. 2000) using the tratransposon piggyBac currently used as a vector has the disadvantage that the recombination efficiency is low and much effort is required to produce the recombinant. is there. In other words, the current method uses a plasmid with the target gene and marker gene inserted between the inverted terminal repeats (IVR) of the transposon piggyBac as a vector, and a plasmid containing a transposon transferase gene called a helper plasmid. It is characterized by being injected into silkworm eggs. That is, it is a method of forcibly expressing a transferase gene in an egg, causing the transfer of the target gene from the vector plasmid to the genome in germ cells, and recovering recombinant individuals in the next generation. The production efficiency of recombinants when crossed between silkworms injected by this method is 0.5-15% when viewed in the number of silkworms where the recombinants appear in all the next-generation injected silkworms. (Imamura et al. 2003; Tomita et al. 2003). This frequency further decreases as the size of the gene to be introduced increases. Therefore, it is required to develop a more efficient recombinant production method.

Prior art document information related to the invention of the present application is shown below.
Imamura, M., J. Nakai, S. Inoue, GX Quan, T. Kanda et al., 2003 Targeted gene expression using the GAL4 / UAS system in the silkworm Bombyx mori. Genetics 165: 1329-1340. Inoue, S., T. Kanda, M. Imamura, GX Quan, K. Kojima et al., 2005 A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins.Insect Biochem Mol Biol 35 : 51-59. Kapetanaki, MG, TG Loukeris, I. Livadaras and C. Savakis, 2002 High frequencies of Minos transposon mobilization are obtained in insects by using in vitro synthesized mRNA as a source of transposase.Nucleic Acids Res 30: 3333-3340. Pavlopoulos, A., AJ Berghammer, M. Averof and M. Klingler, 2004 Efficient transformation of the beetle Tribolium castaneum using the Minos transposable element: quantitative and qualitative analysis of genomic integration events.Genetics 167: 737-746. Tamura, T., C. Thibert, C. Royer, T. Kanda, E. Abraham et al., 2000 Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18: 81-84. Tomita, M., H. Munetsuna, T. Sato, T. Adachi, R. Hino et al., 2003 Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21: 52-56.

  This invention is made | formed in view of such a condition, and the subject which this invention tends to solve is providing the production method of the recombinant of a silkworm with high efficiency.

The present inventors have made various studies in order to solve the above problems. As a result, the transposon piggyBac transferase-encoding mRNA and the transposon piggyBac into which any DNA is inserted are injected into silkworm eggs, and silkworms are produced from the silkworm eggs, thereby improving the production efficiency of transgenic silkworms. It has been found that it can be significantly increased. That is, the present inventors remarkably increase the production efficiency of transgenic silkworms by using in vitro synthesized mRNA instead of the conventionally used helper plasmid as a source of transposon piggyBac transferase. In particular, the present invention has been completed. Specifically, the present invention provides the following [1] to [20].
[1] A method for producing a transgenic silkworm, comprising the following steps (a) and (b):
(A) a step of injecting a silkworm egg with an mRNA having a coding region of a transposon piggyBac transferase and a transposon piggyBac into which an arbitrary DNA has been inserted,
(B) A step of selecting a transgenic silkworm into which an arbitrary DNA has been inserted from silkworms produced from the silkworm egg of (a).
[2] A method for producing a silkworm egg into which an arbitrary DNA has been introduced, comprising a step of injecting an mRNA having a transposon piggyBac transferase coding region and a transposon piggyBac into which an arbitrary DNA has been inserted into a silkworm egg.
[3] A method for introducing arbitrary DNA into a silkworm egg, comprising the step of injecting a transposon piggyBac having a transposon piggyBac transferase coding region into the silkworm egg and a transposon piggyBac into which the arbitrary DNA has been inserted.
[4] The method according to [1] to [3], wherein the mRNA having the transposon piggyBac transferase coding region is mRNA transcribed from the DNA according to any one of (i) to (iv) below: ;
(I) From the 5 ′ untranslated region consisting of the base sequence described in SEQ ID NO: 5, the transposon piggyBac transferase coding region consisting of the base sequence described in SEQ ID NO: 1, and the base sequence described in SEQ ID NO: 10. A DNA having 3 ′ untranslated regions bound in this order,
(Ii) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA having a 3 ′ untranslated region consisting of
(Iii) 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 6, transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and base sequence shown in SEQ ID NO: 10 A DNA having a 3 ′ untranslated region consisting of
(Iv) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 6, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 DNA in which 3 'untranslated regions consisting of are bound in this order.
[5] The method according to [1] to [3], wherein the mRNA having the transposon piggyBac transferase coding region is mRNA transcribed from the DNA of any one of (a) to (i) below: ;
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(G) From the 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 9, the transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are functionally linked in this order;
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA in which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order.
[6] The mRNA according to [1] to [3], wherein the mRNA having the transposon piggyBac transferase coding region is mRNA transcribed from the DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 2 to 4. the method of.
[7] The method according to [1] to [3], wherein the mRNA having the coding region of the transposon piggyBac transferase is mRNA transcribed from a vector comprising the nucleotide sequence of SEQ ID NO: 14 or 15.
[8] The method according to any one of [1] to [7], wherein the transposon piggyBac into which any DNA is inserted has at least the following (a) to (c):
(A) DNA comprising a transposon piggyBac right-hand inverted terminal repeat,
(B) DNA comprising a transposon piggyBac left inverted terminal repeat,
(C) Any DNA sandwiched between (a) and (b) above.
[9] A transgenic silkworm produced by the method according to [1].
[10] A silkworm egg into which an arbitrary gene has been introduced, produced by the method according to [2].
[11] DNA according to any one of (a) to (i) below;
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(G) From the 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 9, the transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA in which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order.
[12] DNA comprising the base sequence set forth in SEQ ID NO: 4.
[13] A vector comprising the base sequence according to [11] or [12].
[14] A vector comprising the base sequence set forth in SEQ ID NO: 15.
[15] A kit comprising a vector comprising the DNA according to any of (i) to (iv) below;
(I) From the 5 ′ untranslated region consisting of the base sequence described in SEQ ID NO: 5, the transposon piggyBac transferase coding region consisting of the base sequence described in SEQ ID NO: 1, and the base sequence described in SEQ ID NO: 10. A DNA having 3 ′ untranslated regions bound in this order,
(Ii) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA having a 3 ′ untranslated region consisting of
(Iii) 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 6, transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and base sequence shown in SEQ ID NO: 10 A DNA having a 3 ′ untranslated region consisting of
(Iv) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 6, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 DNA having a functionally linked 3 ′ untranslated region consisting of
[16] A kit comprising a vector comprising the base sequence set forth in SEQ ID NO: 3.
[17] A kit comprising a vector consisting of the base sequence set forth in SEQ ID NO: 14.
[18] A kit comprising the vector according to [13] or [14].
[19] The kit according to any one of [15] to [18], further comprising a vector having the following (a) to (c):
(A) DNA comprising a transposon piggyBac right-hand inverted terminal repeat,
(B) DNA comprising a transposon piggyBac left inverted terminal repeat,
(C1) DNA containing a cloning site sandwiched between (a) and (b) above.
[20] The kit according to any one of [15] to [18], further comprising a vector having the following (a) to (c):
(A) DNA comprising a transposon piggyBac right-hand inverted terminal repeat,
(B) DNA comprising a transposon piggyBac left inverted terminal repeat,
(C) Any DNA sandwiched between (a) and (b) above.

  The present invention provides a method for producing transgenic silkworms with significantly higher efficiency than the prior art. This has made it possible to significantly reduce the labor required for the production of transgenic silkworms.

  In recent years, attempts have been made to produce recombinant proteins of heterologous organisms in silkworms, which are a kind of insects. The present invention is useful in producing a transgenic silkworm used in such a scene. Among them, it is considered to be particularly useful for the production of recombinant proteins used for test drugs and pharmaceuticals.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a method for producing a recombinant silkworm having high efficiency. The present invention allows the transgenic silkworm to produce a silkworm by injecting a silkworm egg with a transposon into which mRNA encoding a transposon transferase and an arbitrary DNA has been inserted, and producing the silkworm from the silkworm egg. Based on a successful increase in production efficiency. That is, this invention provides the manufacturing method of a transgenic silkworm including the process of the following (a) and (b).
(A) A step of injecting a transposon piggyBac into which a transposon piggyBac transferase coding region is coding and a transposon piggyBac into which an arbitrary DNA has been inserted into a silkworm egg (b) from among silkworms produced from a silkworm egg in (a) For selecting transgenic silkworms with inserted DNA

  In the method for producing a transgenic silkworm of the present invention, first, an mRNA having a transposon piggyBac transferase coding region and a transposon having an arbitrary DNA inserted are injected into a silkworm egg. A transposon is known as a base sequence that can move a position on the genome in a cell, and is often used as a vector for gene transfer. There are two types of transposon: a DNA type in which DNA fragments are directly transferred, and an RNA type that undergoes a process of transcription and reverse transcription. The transposon in the present invention is not limited to either one, and both transposons are included.

  Transposons also require a transferase in order to transfer themselves. The transferase recognizes the inverted terminal repeat of the transposon. This causes the transposon to be excised from the genome sequence and inserted into another site, causing metastasis (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba. M. , Ko ^ moto, N., Thomas, JL, Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, JC, and Couble, P. (2000) A piggyBac-derived vector efficiently promotes germ-line transformation in the silkworm Bombyx mori L. Nature Biotechnology 18, 81-84). The transposon of the present invention is not particularly limited as long as it can be used as a vector for gene transfer, but a preferred transposon is piggyBac. The transposon piggyBac has the highest efficiency as a vector for introducing a gene into a cocoon compared to many other transposons. Therefore, the transposon piggyBac is particularly excellent as a transposon used in the present invention.

  The amino acid sequence of the transferase of the transposon piggyBac of the present invention is shown in SEQ ID NO: 31. The base sequence of DNA encoding the amino acid sequence of piggyBac transferase is shown in SEQ ID NO: 1, and the base sequence of mRNA transcribed from the DNA is shown in SEQ ID NO: 19.

  Here, in the nucleotide sequence of DNA encoding the amino acid sequence of piggyBac transferase, one or more amino acids in the amino acid sequence shown in SEQ ID NO: 31 are substituted, deleted, added, and / or inserted. A DNA encoding a protein having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 31 and the DNA consisting of the base sequence shown in SEQ ID NO: 1 DNA that hybridizes under conditions and that encodes a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 31 is also included. Methods well known to those skilled in the art for isolating such homologous genes include hybridization techniques (Southern, EM, Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR). Technology (Saiki, RK, et al. Science, vol. 230, 1350-1354, 1985, Saiki, RK et al. Science, vol. 239, 487-491, 1988) is known.

  In the present invention, “mRNA having a transposon piggyBac transferase coding region” means an mRNA having at least a 5 ′ untranslated region, a transposon piggyBac transferase coding region, and a 3 ′ untranslated region. That is, the “mRNA having the transposon piggyBac transferase coding region” of the present invention may contain sequences other than the 5 ′ untranslated region, the transposon piggyBac transferase coding region, and the 3 ′ untranslated region. The “mRNA having the transposon piggyBac transferase coding region” of the present invention is a DNA expression cassette that expresses the mRNA of interest, and a vector introduced so that the expression cassette can be expressed. It may be manufactured or chemically synthesized in vitro.

More specifically, the “mRNA having the coding region of the transposon piggyBac transferase” of the present invention is the mRNA transcribed from the DNA according to any one of (i) to (iv) below, Examples include mRNA chemically synthesized based on the sequence information of DNA described in any one of i) to (iv).
(I) From the 5 ′ untranslated region consisting of the base sequence described in SEQ ID NO: 5, the transposon piggyBac transferase coding region consisting of the base sequence described in SEQ ID NO: 1, and the base sequence described in SEQ ID NO: 10. DNA with 3 'untranslated region
(Ii) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. DNA with 3 'untranslated region consisting of
(Iii) 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 6, transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and base sequence shown in SEQ ID NO: 10 DNA with 3 'untranslated region consisting of
(Iv) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 6, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 DNA with 3 'untranslated region consisting of

  In the present invention, as a DNA in which the 5 ′ untranslated region, the transposon piggyBac transferase coding region, and the 3 ′ untranslated region are bound in this order, a particularly preferred embodiment is the base described in SEQ ID NO: 3. Examples include DNA consisting of sequences. When the DNA is synthesized using a biological method, the DNA consisting of the base sequence shown in SEQ ID NO: 3 is contained in the vector consisting of the base sequence shown in SEQ ID NO: 14, MRNA consisting of the base sequence shown in SEQ ID NO: 21 is transcribed from the vector.

  The DNAs (i) to (iv) of the present invention may further have SEQ ID NO: 13 or the sequence (polyA sequence) represented by “n” in SEQ ID NO: 13. . Here, “n” means that “A” is an array repeated x times. In the present invention, x is not limited, but is preferably 90 to 100 (90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100).

Further, as the “mRNA having the transposon piggyBac transferase coding region” in the present invention, the mRNA transcribed from the DNA according to any one of the following (a) to (i), or the following (a) to It is possible to cite mRNA chemically synthesized based on the DNA sequence information described in any of (i).
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(G) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11 A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order

In the present invention, the DNA in which the 5 ′ untranslated region, the transposon piggyBac transferase coding region, the 3 ′ untranslated region, and the polyA sequence are combined in this order is derived from the nucleotide sequence set forth in SEQ ID NO: 4. DNA which becomes. The DNA consisting of the base sequence shown in SEQ ID NO: 4 is contained in the vector consisting of the base sequence shown in SEQ ID NO: 15, from which the mRNA consisting of the base sequence shown in SEQ ID NO: 22 is transcribed. Is done.
In addition, the DNA of said (a)-(i) of this invention does not need to have the base sequence of sequence number: 13. Moreover, you may have the arrangement | sequence (polyA arrangement | sequence) shown by "n" among sequence number: 13.

  In the present invention, the 5 ′ untranslated region has the base sequence WAAWNMAAA (M is A or C; N is A, U, G or C; W is U or A, SEQ ID NO: 7) or TAAAATAAA (SEQ ID NO: It is preferable to have 8). This sequence is a sequence commonly found upstream of the translation start point in a known silkworm gene. In particular, the base sequence TAAAACAAA (SEQ ID NO: 8) is important for protein synthesis in silkworm because it exists upstream of the translation start point of β-tubulin gene, which is known to be expressed in a large amount in silkworm cells. Presumed to be an array. The mRNA having the coding region of the transposon piggyBac transferase of the present invention preferably has such a sequence.

  On the other hand, for sequences other than TAAAACAAA (ie, GAATACTCAAGCTAGGG sequence) in the 5 ′ untranslated region (base sequence described in SEQ ID NO: 9), the GAATACTCAAGCTAGGG sequence, for example, when a part of the base of the GAATACTCAAGCTAGGG sequence is substituted. A partly different arrangement may be used. In particular, when the mRNA of the present invention is prepared using a vector, it is designed in combination with a promoter. A person skilled in the art can appropriately design a sequence other than TAAAACAAA according to the promoter sequence.

  The mRNA having the coding region of the transposon piggyBac transferase of the present invention may be produced in any manner. The mRNA having the coding region of the transposon piggyBac transferase of the present invention can be produced by any method known to those skilled in the art. For example, an expression cassette for DNA that expresses the target mRNA is prepared. Examples of the method include a method of producing using a vector introduced so that the expression cassette can be expressed, a method of chemically synthesizing in vitro, and the like. However, the method for producing mRNA having the coding region of the transposon piggyBac transferase of the present invention is not limited to these, and any method known to those skilled in the art can be used.

When the mRNA of the present invention is biologically synthesized using eukaryotic cells, a “polyA region” is added to the 3 ′ untranslated region of the synthesized product. The “mRNA having the coding region of the transposon piggyBac transferase” in the present invention also includes an mRNA to which a “polyA region” has been added when transcribed into mRNA.
On the other hand, when the mRNA of the present invention is chemically synthesized using an in vitro synthesis system, addition of the “polyA region” does not occur. The “mRNA having the transposon piggyBac transferase coding region” in the present invention includes an mRNA having no “polyA region” as described above.

  As described above, the DNA of the present invention may have any other sequence as long as it has a 5 ′ untranslated region, a transposon piggyBac transferase coding region, and a 3 ′ untranslated region. Good. Thus, for example, an arbitrary sequence may be present between the 5 ′ untranslated region and the transposon piggyBac transferase coding region, and between the transposon piggyBac transferase coding region and the 3 ′ untranslated region. May have any sequence. Furthermore, it may have an arbitrary sequence further on the 5 'end side of the 5' untranslated region, and may have an arbitrary sequence further on the 3 'end side of the 3' untranslated region.

  In particular, when the DNA of the present invention is synthesized biologically, the DNA of the present invention comprises a 5 ′ untranslated region, a transposon piggyBac transferase coding region, and a 3 ′ untranslated region, or a 5 ′ untranslated region. A transposon piggyBac transferase coding region, a 3 ′ untranslated region, and a polyA sequence, and the mRNA is transcribed and expressed when mRNA containing the DNA is inserted into the vector and expressed from the vector As long as it is, it is not limited. Therefore, when the DNA of the present invention is biologically synthesized, the DNA of the present invention can be used even in the case where an arbitrary sequence is present between, for example, the 5 ′ untranslated region and the transposon piggyBac transferase coding region. The inclusion is as described above.

  In addition, by adding a 5 'untranslated region, 3' untranslated region, and / or polyA sequence to the mRNA sequence encoding the transposon piggyBac transferase, the efficiency of introducing the DNA inserted into the transposon into the silkworm body is increased. It becomes possible to make it.

  In addition, the 5 'untranslated region and / or the 3' untranslated region used in the present invention is not particularly limited. The 5 ′ untranslated region and / or 3 ′ untranslated region of the present invention uses a 5 ′ untranslated region and / or a 3 ′ untranslated region derived from the same species as the organism from which the transposon piggyBac transferase is derived. Of course, it is also possible to use 5 'untranslated regions and / or 3' untranslated regions from different organisms. In addition, the 5 ′ untranslated region and / or the 3 ′ untranslated region of the transposon piggyBac transferase may be used, or the 5 ′ untranslated region and / or the 3 ′ untranslated region derived from mRNA encoding other proteins. It is also possible to use areas. The 5 ′ untranslated region and / or 3 ′ untranslated region used in the method of the present invention is a 5 ′ untranslated region and / or 3 ′ untranslated region naturally present in the mRNA encoding the transferase used. It is preferable. For example, when a transposon piggyBac transferase (a protein consisting of the amino acid sequence described in SEQ ID NO: 31) is used as a transferase, the 5 ′ untranslated region and 3 ′ untranslated region used in combination therewith are: It is preferred that the mRNA encoding the transposon piggyBac transferase is naturally occurring. The 5 ′ untranslated region and 3 ′ untranslated region naturally contained in the mRNA encoding the transposon piggyBac transferase include the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5 and SEQ ID NO: 10. The 3 'untranslated region which consists of a description of a base sequence can be mentioned. Further, the polyA sequence used in the present invention is not particularly limited as described above.

In the method for producing a transgenic silkworm of the present invention, a transposon piggyBac into which an arbitrary DNA has been inserted is also injected into a silkworm egg together with mRNA having a transposon piggyBac transferase coding region. The transposon piggyBac into which any DNA of the present invention is inserted has at least the following (a) to (c).
(A) DNA containing transposon piggyBac right-hand inverted terminal repeat sequence
(B) DNA containing the transposon piggyBac left inverted terminal repeat sequence
(C) Arbitrary DNA sandwiched between (a) and (b) above

  In the present invention, the DNA inserted into the transposon piggyBac is not particularly limited, and any gene or functional DNA can be inserted. Examples of arbitrary genes include kynurenine oxidase gene and DsRed. Examples of functional DNA include DNA encoding RNA having an antisense effect and DNA encoding RNA having an RNAi effect. Examples of the arbitrary DNA in the method of the present invention include such a DNA, but the present invention is not limited thereto, and any DNA can be used.

  As a method for injecting a transposon piggyBac having a transposon piggyBac transferase coding region and a transposon piggyBac into which arbitrary DNA has been inserted into a silkworm egg, for example, using a tube for DNA injection into a silkworm egg directly in the egg Examples include a method of introducing mRNA having the coding region of the transposon piggyBac transferase and a transposon piggyBac into which an arbitrary DNA has been inserted. Further, as a preferred embodiment, there is a method in which a hole is formed in an eggshell physically or chemically in advance, and an mRNA having a transposon piggyBac transferase coding region and a transposon piggyBac into which an arbitrary DNA is inserted are introduced from the hole. Can be mentioned. In particular, at this time, the injection tube is preferably inserted into the egg through the hole so that the insertion angle is substantially perpendicular to the ventral side surface of the egg.

  In the present invention, as a method for physically making a hole in an eggshell, for example, a method for making a hole using a needle, a micro laser, or the like can be mentioned. Preferably, the eggshell can be pierced by a method using a needle. As long as the needle can make a hole in the eggshell of a silkworm, the material, strength, etc. of the needle are not particularly limited. The needle in the present invention usually refers to a rod-like needle having a sharp tip, but is not limited to this shape, and the overall shape is not particularly limited as long as it can make a hole in the eggshell. For example, a pyramidal material with a sharp tip or a triangular pyramid shaped material with a sharp tip is also included in the “needle” of the present invention. In the present invention, a tungsten needle can be preferably used. The thickness (diameter) of the needle of the present invention may be a thickness that can open a hole through which a capillary described later can be made, and is usually 2 to 20 μm, preferably 5 to 10 μm. On the other hand, as a method of chemically making a hole in an eggshell, for example, a method of making a hole using a chemical (such as hypochlorous acid) can be mentioned.

  In the present invention, the position where the hole is made is not particularly limited as long as the insertion angle with respect to the side surface on the ventral side of the egg can be made almost vertical when an injection tube is inserted through the hole. It is the side surface on the side or the opposite side, more preferably the ventral side surface, and still more preferably the middle part of the egg on the ventral side surface slightly from the rear end.

  In the present invention, “substantially vertical” means 70 ° to 120 °, preferably 80 ° to 90 °. The material, strength, inner diameter, etc. of the tube for injection of the present invention are not particularly limited, but if the hole is physically or chemically opened before inserting the tube for injection, the tube is opened. It is preferable that the thickness (outer diameter) can pass through the hole. Examples of the injection tube of the present invention include a glass capillary.

  In the method of introducing the transposon piggyBac into which the mRNA having the coding region of the transposon piggyBac transferase of the present invention and any DNA is inserted, a preferred embodiment is to physically or chemically make a hole in the silkworm egg and inject it. A tube for insertion is inserted into the egg through the hole so that the insertion angle is substantially perpendicular to the ventral side surface of the egg, and mRNA having a transposon piggyBac transferase coding region and any DNA are inserted. The step of injecting the produced transposon piggyBac is performed using a manipulator in which a needle and an injection tube are integrated. In general, the present invention is preferably implemented using an apparatus having the manipulator as one of the components.

  Such devices include a dissecting microscope, an illumination device, a movable stage, a coarse manipulator fixed to a microscope with a metal fitting, a micromanipulator attached to this manipulator, and a transposon piggyBac transferase coding region. And an injector for adjusting the air pressure for injecting a transposon piggyBac into which an arbitrary DNA has been inserted. The pressure used for the injector is supplied from a nitrogen cylinder, and the pressure switch can be turned on by a foot switch. Injection is performed on an egg fixed on a substrate such as a glass slide, and the position of the egg is determined by a movable stage. The glass capillaries of the micromanipulator are operated by an operation unit connected by four tubes. The actual procedure is to determine the position of the tungsten needle relative to the egg with a coarse manipulator, and move the egg horizontally with the stage lever to make a hole. Subsequently, the lever of the operation part of the micromanipulator is operated to guide the tip of the glass capillary to the position of the hole, and the capillary is again inserted into the egg by the lever of the stage. In this case, it is necessary to insert a glass capillary perpendicular to the ventral side surface of the egg. A foot switch is turned on to inject mRNA having a transposon piggyBac transferase coding region and transposon piggyBac into which arbitrary DNA has been inserted, and the lever is operated to pull the capillary from the egg. The hole is covered with an instant adhesive and protected with an incubator with a constant temperature and a constant humidity. The apparatus used in the present invention preferably includes the apparatus described in Japanese Patent No. 1654050 or an apparatus obtained by improving the apparatus.

  In the embodiment of the present invention, it is preferable that the silkworm egg into which the mRNA having the transposon piggyBac transferase coding region and the transposon piggyBac into which an arbitrary DNA has been introduced is introduced are immobilized on the substrate. As the substrate of the present invention, for example, a slide glass, a plastic plate or the like can be used, but is not particularly limited thereto. In the above embodiment of the present invention, in order to inject mRNA having a transposon piggyBac transferase coding region accurately at a position that will become a germ cell in the silkworm egg in the future, and a transposon piggyBac into which any DNA is inserted. It is desirable to fix the eggs in the same direction. Moreover, in the said aspect, there is no restriction | limiting in particular in the number of the silkworm eggs fixed to a board | substrate. When a plurality of silkworm eggs are used, the directionality for fixing the silkworm eggs to the substrate is preferably such that the orientation of the dorsal belly is constant. The silkworm egg is fixed to the substrate of the present invention by, for example, laying eggs on a commercially available mount (rose seed mount) pre-applied with aqueous glue, adding water to the mount to peel off the egg, and then getting wet The eggs are aligned on a substrate and air-dried. The eggs are preferably fixed on the slide glass with the direction of the eggs aligned. Also, the egg can be fixed to the base by using a double-sided tape or an adhesive.

  In addition, as silkworms in the present invention, silkworms having the property of producing non-dormant eggs, silkworms having the property of producing dormant eggs (for example, the practical varieties Gunma, 200, Shunchu, Kangetsu, Nishikiaki, Kanwa, etc.) can be used. Here, a dormant egg refers to an egg in which embryonic development stops temporarily after spawning, and a non-diapause egg refers to an egg in which postnatal embryonic development does not stop and larvae hatch.

  When using a silkworm having the property of producing a dormant egg, a non-dormant egg is laid, and an mRNA having a transposon piggyBac transferase coding region and a transposon piggyBac into which any DNA is inserted are inserted into the non-dormant egg. Introduce. For example, in Gunma, the method of allowing non-dormant eggs to lay on adults produced from the dormant eggs by culturing the dormant eggs at 15 ° C. to 21 ° C. Is cultured at 16 ° C. to 20 ° C. to allow the adults generated from the dormant eggs to produce non-dormant eggs, and more preferably, the dormant eggs are cultured at 18 ° C. so that the adults generated from the dormant eggs are non-destructive. A method of giving birth to a dormant egg, most preferably a method in which a larva produced from a dormant egg is cultivated at 18 ° C., and then a non-dormant egg is given to a grown adult. Can do. In addition, in 200, a method in which a dormant egg is laid at 15 ° C. to 21 ° C. to allow an adult produced from the dormant egg to produce a non-dormant egg, preferably the dormant egg is cultured at 16 ° C. to 20 ° C. A method of allowing non-dormant eggs to lay on the adults generated from the dormant eggs, and more preferably a method of causing non-dormant eggs to lay on the adults generated from the dormant eggs by culturing the dormant eggs at 18 ° C. A method of raising larvae generated from eggs in full light and allowing grown adults to lay non-dormant eggs, most preferably culturing dormant eggs at 25 ° C. to raise larvae generated from dormant eggs in full light And a method of causing the grown adult to lay non-dormant eggs.

  Egg culturing can be performed, for example, by placing it in an incubator at 18 ° C. to 25 ° C. or a constant temperature room, and raising larvae can be performed using artificial feed in a breeding room at 20 ° C. to 29 ° C. .

  Those skilled in the art can culture the dormant egg of the present invention according to a general silkworm egg culture method. For example, the culture is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture.pp193, Jikkyo Publishing, Tokyo”. In addition, breeding of silkworm larvae in the present invention can be performed by those skilled in the art by a well-known method. For example, breeding is performed according to the method described in “Ministry of Education (1978) Soybean Manufacture.pp193, Jikkyo Publishing, Tokyo”.

  In the present invention, whether or not a laid egg is a non-dormant egg can be determined by the color of the egg. In general, it is known that dormant eggs are colored dark brown and non-dormant eggs are yellowish white. Therefore, in the present invention, an egg laid is determined to be a non-dormant egg by not being dark brown, more preferably yellowish white.

  Next, in the method for producing a transgenic silkworm of the present invention, mRNA having a transposon piggyBac transferase coding region and arbitrary DNA were inserted from the silkworms produced from the silkworm eggs obtained as described above. A transgenic silkworm having a transposon piggyBac inserted therein is selected. Whether or not an mRNA having a transposon piggyBac transferase coding region and a transposon piggyBac into which an arbitrary DNA has been introduced into a silkworm egg, for example, an mRNA having an injected transposon piggyBac transferase coding region, and Method of re-extracting and measuring transposon piggyBac with any DNA inserted (Nagaraju, J., Kanda, T., Yukuhiro, K., Chavancy, G., Tamura, T. and Couble, P (1996) Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection of foreign DNA. Appl. Entomol. Zool., 31, 589-598) and the transposon piggyBac transferase coding region injected How to see mRNA expression in eggs (Tamura, T., Kanda, T., Takiya, S., Okano, K. and Maekawa, H. (1990). Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm, Bombyx mori. Jpn. J. Genet., 65, 401-410) It is possible to confirm me.

  The present invention also relates to a method for producing a silkworm egg into which an arbitrary DNA has been introduced, which comprises a step of injecting an mRNA having a transposon piggyBac transferase coding region and a transposon having an arbitrary DNA inserted into the silkworm egg. The present invention further relates to a method for introducing an arbitrary DNA into a silkworm egg, comprising the step of injecting an mRNA having a transposon piggyBac transferase coding region and a transposon into which the arbitrary DNA has been inserted into a silkworm egg.

  MRNA having a transposon piggyBac transferase coding region used in the method for producing a transgenic silkworm of the present invention and / or silkworm egg, 5 ′ untranslated region, transposon piggyBac transferase, 3 ′ untranslated region , PolyA sequence, arbitrary DNA and the like are as described above. In addition, it is also possible to inject mRNA having a transposon piggyBac transferase coding sequence and a transposon into which an arbitrary gene has been inserted into a silkworm egg by the above-described method.

  The present invention also provides a transgenic silkworm produced by the method for producing a transgenic silkworm of the present invention, and a silkworm egg produced by the method for producing a silkworm egg of the present invention. Since these transgenic silkworms and silkworm eggs can be used for the production of recombinant proteins, they are useful in the field of production of useful substances.

The present invention further provides DNA in which a 5 ′ untranslated region, a transposon piggyBac transferase coding region, a 3 ′ untranslated region, and a polyA sequence are functionally linked. More specifically, the present invention provides the DNA described in any of (a) to (i) below.
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(G) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11 A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order

  In the present invention, as a DNA in which the 5 ′ untranslated region, the transposon piggyBac transferase coding region, and the 3 ′ untranslated region are bound in this order, a particularly preferred embodiment is the base described in SEQ ID NO: 3. Examples include DNA consisting of sequences. DNA consisting of the base sequence shown in SEQ ID NO: 3 is contained in a vector consisting of the base sequence shown in SEQ ID NO: 14, and mRNA consisting of the base sequence shown in SEQ ID NO: 21 is transcribed from the vector. Is done.

  As described above, the DNAs (a) to (d) of the present invention further have SEQ ID NO: 13 or the sequence indicated by “n” in SEQ ID NO: 13 (polyA sequence). You may do it. Here, “n” means that “A” is an array repeated x times. In the present invention, x is not limited, but is preferably 90 to 100 (90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100).

  MRNA generated from such DNA can be used in the method for producing a transgenic silkworm of the present invention. It can also be used in a method for producing silkworm eggs, a method for introducing arbitrary DNA into silkworm eggs, and the like. The DNA of the present invention can also be used as a kit used in these methods.

  The present invention also provides a vector comprising the DNA according to any one of (a) to (i) above. In the present invention, as a vector containing the DNA described in any one of (a) to (i) above, a preferable embodiment includes DNA comprising the base sequence described in SEQ ID NO: 4. A particularly preferred embodiment is a vector consisting of the base sequence set forth in SEQ ID NO: 15. The vector of the present invention is useful for synthesizing mRNA having the transposon piggyBac transferase coding region. It can also be used as a kit for use in the method of the present invention.

  Examples of the promoter functionally linked to the expression cassette of the present invention include SP6 promoter and T7 promoter, but the promoter used in the present invention is not limited to these and expresses the expression cassette of the present invention. Any promoter can be used as long as it is possible.

The present invention further provides a kit comprising a vector containing the DNA according to any one of (i) to (iv) below.
(I) From the 5 ′ untranslated region consisting of the base sequence described in SEQ ID NO: 5, the transposon piggyBac transferase coding region consisting of the base sequence described in SEQ ID NO: 1, and the base sequence described in SEQ ID NO: 10. DNA with 3 'untranslated region
(Ii) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. DNA with 3 'untranslated region consisting of
(Iii) 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 6, transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and base sequence shown in SEQ ID NO: 10 DNA with 3 'untranslated region consisting of
(Iv) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 6, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 DNA with 3 'untranslated region consisting of

Specifically, the present invention provides:
1) A kit comprising a vector comprising the DNA according to any one of (i) to (iv) above and a vector having at least the following (a) to (c1) is provided.
(A) DNA containing transposon piggyBac right-hand inverted terminal repeat sequence
(B) DNA containing the transposon piggyBac left inverted terminal repeat sequence
(C1) DNA containing a cloning site sandwiched between the above (a) and (b)

2) A kit comprising a vector comprising the DNA according to any one of (i) to (iv) above and a vector having at least the following (a) to (c2) is provided.
(A) DNA containing transposon piggyBac right-hand inverted terminal repeat sequence
(B) DNA containing the transposon piggyBac left inverted terminal repeat sequence
(C2) Arbitrary DNA sandwiched between (a) and (b) above

The present invention further provides a kit comprising a vector containing the DNA according to any one of (a) to (i) below.
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A 3 'untranslated region and DNA having the base sequence set forth in SEQ ID NO: 13 bound in this order
(G) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11 A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA comprising a 3 ′ untranslated region and a base sequence set forth in SEQ ID NO: 13 in this order

Specifically, the present invention provides:
3) A kit comprising a vector comprising the DNA according to any one of (a) to (i) above and a vector having at least the following (a) to (c1) is provided.
(A) DNA containing transposon piggyBac right-hand inverted terminal repeat sequence
(B) DNA containing the transposon piggyBac left inverted terminal repeat sequence
(C1) DNA containing a cloning site sandwiched between the above (a) and (b)

4) A kit comprising a vector comprising the DNA of any one of (a) to (i) above and a vector having at least the following (a) to (c2) is provided.
(A) DNA containing transposon piggyBac right-hand inverted terminal repeat sequence
(B) DNA containing the transposon piggyBac left inverted terminal repeat sequence
(C2) Arbitrary DNA sandwiched between (a) and (b) above

  Examples of the basic skeleton of the vector having at least (a) to (c1) or (a) to (c2) include the basic skeleton of piggyBac.

The present invention also provides a kit comprising mRNA encoding a transposon transferase, which is used to inject an arbitrary gene into a silkworm egg. The explanation of the mRNA encoding the transposon transferase is as described above. Such a kit can contain any DNA
Can be used to inject silkworm eggs.

  In addition, sequence number: 19-24 of the sequence table of this specification is the base sequence of mRNA transcribed from DNA which consists of a base sequence of sequence number: 1-6 of the sequence table of this specification, respectively. . In addition, SEQ ID NOs: 25 to 30 in the sequence listing of the present specification are the base sequences of mRNA transcribed from the DNA comprising the base sequences described in SEQ ID NOs: 8 to 13 of the sequence listing of the present specification, respectively. .

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

1. The w1pnd strain, which is non-dormant with white eyes and white eggs, was used for the production of strains and reared recombinant silkworms. Silkworms were bred at 25 ° C. using artificial feed (Nippon Agricultural Industry, for original species).
2. Plasmid for mRNA synthesis

The plasmid pBSII-IFP2-orf (FIG. 1A, SEQ ID NO: 14) was obtained from M. Frazer ( http://piggybac.bio.nd.edu/ ). Moreover, the plasmid pGEMe-pigORF-p (A) 90 (FIG. 1B, SEQ ID NO: 12) was prepared by the following method (FIG. 2). That is, using plasmid pHA3PIG (Tamura et al. 2000) as a template, two kinds of primers, 5'-CCCGGGTAAAACAAAATGGGATGTT-3 '(primer 1, SEQ ID NO: 17) and 5'-ACTAGTTGTTTATTTATTTATTAAAAAA-3' (primer 2, SEQ ID NO: : 18) was used to perform PCR with TaKaRa LA Taq (TaKaRa) to amplify the ORF portion of piggyBac transferase. PCR reaction conditions were 96 ° C for 10 seconds, 94 ° C for 5 seconds, 55 ° C for 30 seconds, 68 ° C for 3 minutes for 5 cycles, 94 ° C for 1 minute, 55 ° C for 10 seconds, 68 ° C for 2 minutes for 25 cycles, Then 68 ° C for 5 minutes. The DNA fragment amplified by PCR was directly cloned into the plasmid pGEM-T Easy (Promega). A DNA fragment excised from this plasmid with restriction enzymes EcoR I and Bgl II, a DNA fragment of about 600 bp on the 3 ′ side, also excised with Pst I and Spe I, and a restriction enzyme Bgl II from the plasmid pHA3PIG (Tamura et al. 2000). The plasmid pBS-pigORF was prepared by incorporating the DNA fragment excised by treatment with Pst I between the EcoR I and Spe I sites of the plasmid pBluescriptII SK- (Stratagene). Next, 30 mer oligodT and 30 mer oligodA were used as primers and templates for 1 minute treatment at 94 ° C, followed by 30 cycles of 94 ° C for 1 second, 55 ° C for 5 seconds, 72 ° C, 90 seconds, 72 ° C for 2 minutes The reaction product DNA was precipitated with ethanol. The precipitated DNA was dissolved in distilled water, the ends of the DNA were smoothed with T4 polymerase, and ligation was performed. Next, A-tail was added by reacting the obtained DNA with Ex-Taq polymerase (TaKaRa) at 72 ° C. for 10 minutes, and this was cloned into plasmid pGEM-T Easy (Promega) to obtain plasmid pGEMe-p. (A) Created 90. From the results of sequencing and agarose electrophoresis, the fragment inserted into the constructed plasmid was confirmed to be a poly A sequence of about 90 bp. Next, the plasmid pGEMe-p (A) 90 was digested with EcoT22 I, smoothed with T4 polymerase, and further digested with the restriction enzyme Spe I. The digested plasmid DNA is treated with the restriction plasmids Sma I and Spe I and the DNA fragment containing the ORF of the piggyBac transferase to be excised is incorporated into the plasmid DNA after digestion to obtain plasmid pGEMe-pigORF-p ( A) 90 was produced.

Here, the plasmid of FIG. 2 has two contrivances in order to efficiently produce a transferase from mRNA in the silkworm organism.
The first contrivance is the base sequence upstream of the translation start point. As a result of comparing the sequences upstream of the transcription start point for the already known silkworm genes, the base sequence WAAWNMAAA (M is A or C; N is A, T, G or C; W is T or A, SEQ ID NO: 7) was found to exist in each gene as a common sequence. From this, it was speculated that this common sequence is an important sequence for synthesizing proteins from mRNA in silkworms. The β-tubulin gene, which is known to have a high expression level in silkworm cells, had TAAAACAAA (SEQ ID NO: 8) corresponding to this common sequence in the upstream region of the translation start point. Therefore, a gene was synthesized so that TAAAACAAA of this β-tubulin gene would be the upstream region sequence of the translation start point of the transposon piggyBac transferase gene, and a plasmid for synthesizing mRNA having this sequence was prepared (FIG. 2).

The second contrivance is to insert a sequence for adding a poly (A) fragment downstream of the 3 ′ UTR for the purpose of stabilizing mRNA. This sequence was synthesized into a poly (A) fragment by PCR and inserted into a plasmid (FIG. 2).
3. mRNA synthesis in vitro

The plasmids pBSII-IFP2-orf and pGEMe-pigORF-p (A) 90 were purified using the QIAprep Spin Miniprep Kit (QIAGEN). 4 μg of the purified plasmid DNA was digested with restriction enzyme Not I, treated with phenol / chloroform and then ethanol precipitated, and the precipitate was dissolved in a buffer of 10 mM Tris (pH 8.0) / 1 mM EDTA. Using 1 μg of this as a template, mRNA was synthesized in vitro using mMESSAGE mMACHINE ^™ (Ambion) and purified. After quantifying the mRNA in the obtained aqueous solution, ethanol precipitation was performed and dissolved in a buffer solution for injection. Storage was performed at -80 ° C.
4). Production of recombinant silkworm

  Recombinant silkworms were produced by injecting a mixture of vector and mRNA into eggs immediately after laying (Tamura et al. 2000). The vector was 200 μg / ml pBac3xP3DsRed (Inoue et al. 2005); FIG. 1D, SEQ ID NO: 13), and the helper mRNA was pBSII-IFP2-orf or pGEMe-pigORF-p (A ) MRNA synthesized from 90 was used. For comparison, a conventional helper plasmid pHA3PIG mixed with a vector plasmid (200 μg / ml) at a concentration of 200 μg / ml was used. The eggs were injected according to Tamura et al. (2000). Recombinant silkworms were screened by the method of Inoue et al. (Inoue et al. 2005).

  The method for producing the recombinant silkworm is shown in FIG. That is, these plasmids are purified and cleaved with the restriction enzyme Not I to produce linear DNA. By using a commercially available in vitro mRNA synthesis kit using this DNA as a template, the transposon piggyBac transferase mRNA can be obtained. Obtained. Moreover, it refine | purified with the commercially available purification kit about a vector or a helper plasmid, and produced the solution for injection. The prepared three kinds of solutions were injected into eggs in the early stages of silkworm development, and the appearance rate of recombinant individuals in the next generation was examined by the expression of DsRed (FIG. 3). As a result, as shown in Table 1, when mRNA synthesized using plasmid pBSII-IFP-orf2 as a template was used as a helper, 26% of female pups laid eggs containing recombinants. When mRNA synthesized using the newly produced plasmid pGEMe-pigORF-p (A) 90 as a template was used, 44% of female pups laid eggs containing recombinant individuals (Table 1). On the other hand, as a result of conducting the same experiment using a conventional helper plasmid, as before, 11.6% of female pups laid eggs containing recombinant silkworms. These results indicate that the use of transferase mRNA instead of plasmid DNA as a helper increases the production efficiency of recombinant silkworms. Compared to the case where plasmid DNA was used as a helper, the newly generated plasmid pGEMe-pigORF-p (A) 90 was approximately doubled when mRNA synthesized using plasmid pBSII-IFP2-orf was used as a template. It was found that the efficiency was about 4 times higher when used.

* 1: An injection solution consisting of vector DNA (200 ng / μl) and synthetic mRNA (100 ng / μl) was used.
* 2: An injection solution consisting of vector DNA (200 ng / μl) and helper plasmid DNA, pHA3PIG (200 ng / μl) was used.

  As a similar example, it has been reported that Minos, which is a transposon other than piggyBac (a transposon of a completely different genus), uses transferase mRNA instead of plasmid DNA to increase the efficiency of recombinant production. Yes. In an example using Minos, a 2 to several fold improvement in efficiency was reported by injecting Minos transferase mRNA into eggs instead of helper plasmids (Kapetanaki et al. 2002; Pavlopoulos et al. 2004).

To date, attempts have been made to increase the efficiency of production of recombinant individuals by using mRNA instead of the helper plasmid in the transposon piggyBac. However, no efficiency has been improved, and this is the first example. In this experiment, we added a gene sequence that is expressed in large quantities in silkworm upstream of the translation start point of mRNA, created a plasmid that forcibly attaches polyA sequence to the 3 'end, and using this mRNA, It turns out that efficiency goes up. From these facts, it is concluded that the method newly developed in this experiment is effective as an efficient method for producing a recombinant silkworm.

It is a figure which shows the structure of the plasmid used for production of a recombinant silkworm. A to D are respectively A; a plasmid used for mRNA synthesis (pBSII-IFP2-orf), B; a newly created plasmid for mRNA synthesis (pGEMe-pigORF-p (A) 90), C; a helper plasmid ( pigA3helper), D; vector plasmid (pBac3xP3DsRed). It is a figure which shows the flowchart of the production process of plasmid pGEMe-pigORF-p (A) 90. In the figure, (a) is a step of inserting a DNA fragment amplified by primers 1 and 2 into plasmid pGEM-T Easy, (b) is a step of inserting into plasmid pGEM-T Easy, (c) is a plasmid pBluescript. It means the process of inserting. (A) It is a figure which shows the production method of the recombinant silkworm using mRNA. (B) A photograph showing a recombinant produced by the method described in FIG.

Claims (20)

A method for producing a transgenic silkworm, comprising the following steps (a) and (b):
(A) a step of injecting a silkworm egg with an mRNA having a coding region of a transposon piggyBac transferase and a transposon piggyBac into which an arbitrary DNA has been inserted,
(B) A step of selecting a transgenic silkworm into which an arbitrary DNA has been inserted from silkworms produced from the silkworm egg of (a). A method for producing a silkworm egg into which an arbitrary DNA has been introduced, comprising a step of injecting an mRNA having a transposon piggyBac transferase coding region and a transposon piggyBac into which an arbitrary DNA has been inserted into a silkworm egg. A method of introducing arbitrary DNA into a silkworm egg, comprising a step of injecting an mRNA having a coding region of a transposon piggyBac transferase and a transposon piggyBac into which arbitrary DNA has been inserted into a silkworm egg. The method according to claim 1, wherein the mRNA having a transposon piggyBac transferase coding region is mRNA transcribed from the DNA of any one of (i) to (iv) below:
(I) From the 5 ′ untranslated region consisting of the base sequence described in SEQ ID NO: 5, the transposon piggyBac transferase coding region consisting of the base sequence described in SEQ ID NO: 1, and the base sequence described in SEQ ID NO: 10. A DNA having 3 ′ untranslated regions bound in this order,
(Ii) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA having a 3 ′ untranslated region consisting of
(Iii) 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 6, transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and base sequence shown in SEQ ID NO: 10 A DNA having a 3 ′ untranslated region consisting of
(Iv) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 6, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 DNA in which 3 'untranslated regions consisting of are bound in this order. The method according to claims 1 to 3, wherein the mRNA having a transposon piggyBac transferase coding region is mRNA transcribed from the DNA of any one of (a) to (i) below;
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(G) From the 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 9, the transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are functionally linked in this order;
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA in which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order. The method according to claim 1, wherein the mRNA having the transposon piggyBac transferase coding region is mRNA transcribed from DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 2 to 4. The method according to claim 1, wherein the mRNA having the transposon piggyBac transferase coding region is mRNA transcribed from a vector comprising the nucleotide sequence of SEQ ID NO: 14 or 15. The method according to any one of claims 1 to 7, wherein the transposon piggyBac into which any DNA is inserted has at least the following (a) to (c):
(A) DNA comprising a transposon piggyBac right-hand inverted terminal repeat,
(B) DNA comprising a transposon piggyBac left inverted terminal repeat,
(C) Any DNA sandwiched between (a) and (b) above. A transgenic silkworm produced by the method according to claim 1. A silkworm egg into which an arbitrary gene is introduced, produced by the method according to claim 2. DNA according to any one of (a) to (i) below;
(A) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(B) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(C) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 7, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(D) From the 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 8, the transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(E) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(F) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 8, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 12 A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(G) From the 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 9, the transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 10. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(H) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA to which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order;
(I) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 9, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 12. A DNA in which the 3 ′ untranslated region and the nucleotide sequence set forth in SEQ ID NO: 13 are bound in this order. DNA consisting of the base sequence set forth in SEQ ID NO: 4. A vector comprising the base sequence according to claim 11 or 12. A vector comprising the nucleotide sequence set forth in SEQ ID NO: 15. A kit comprising a vector comprising the DNA of any one of (i) to (iv) below;
(I) From the 5 ′ untranslated region consisting of the base sequence described in SEQ ID NO: 5, the transposon piggyBac transferase coding region consisting of the base sequence described in SEQ ID NO: 1, and the base sequence described in SEQ ID NO: 10. A DNA having 3 ′ untranslated regions bound in this order,
(Ii) a 5 ′ untranslated region comprising the nucleotide sequence set forth in SEQ ID NO: 5, a transposon piggyBac transferase coding region comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence set forth in SEQ ID NO: 11. A DNA having a 3 ′ untranslated region consisting of
(Iii) 5 ′ untranslated region consisting of the base sequence shown in SEQ ID NO: 6, transposon piggyBac transferase coding region consisting of the base sequence shown in SEQ ID NO: 1, and base sequence shown in SEQ ID NO: 10 A DNA having a 3 ′ untranslated region consisting of
(Iv) a 5 ′ untranslated region comprising the base sequence set forth in SEQ ID NO: 6, a transposon piggyBac transferase coding region comprising the base sequence set forth in SEQ ID NO: 1, and the base sequence set forth in SEQ ID NO: 11 DNA having a functionally linked 3 ′ untranslated region consisting of A kit comprising a vector comprising the base sequence set forth in SEQ ID NO: 3. A kit comprising a vector comprising the nucleotide sequence set forth in SEQ ID NO: 14. A kit comprising the vector according to claim 13 or 14. Furthermore, the kit in any one of Claims 15-18 containing the vector which has the following (a)-(c);
(A) DNA comprising a transposon piggyBac right-hand inverted terminal repeat,
(B) DNA comprising a transposon piggyBac left inverted terminal repeat,
(C) DNA containing a cloning site sandwiched between (a) and (b) above. Furthermore, the kit in any one of Claims 15-18 containing the vector which has the following (a)-(c);
(A) DNA comprising a transposon piggyBac right-hand inverted terminal repeat,
(B) DNA comprising a transposon piggyBac left inverted terminal repeat,
(C) Any DNA sandwiched between (a) and (b) above.