JP2019103471A - Gene-transfer vector for mammalian cells - Google Patents

Abstract

To provide gene introduction means capable of introducing a gene larger than before into non-human animal cells, and making the acquisition rate of transgenic animals close to 100%, for the purpose of creating transgenic non-human model animals of genes related to advanced functions.SOLUTION: Using a piggyBac transposase and a piggyBac transposon vector to which a transposon region comprising a foreign protein promoter DNA that functions in mammalian cells and a DNA encoding a foreign protein operably linked downstream of the promoter DNA as well as a DNA encoding fluorescent protein for determination at downstream of the promoter DNA for determination functioning in mammalian cells and upstream of an ITR sequence are operably linked, the DNA encoding the foreign protein is introduced into a genomic DNA of a host.SELECTED DRAWING: None

Description

  The present invention relates to a piggyBac transposon vector for foreign gene transfer into mammalian cells, and according to the present invention, a gene is introduced into a non-human mammalian early embryo or the like by using the vector and piggyBac transposase. Thus, transgenic non-human mammals that can be used as human disease model animals can be produced, and foreign transgenic cells for screening can be prepared.

  The gene transfer method is an important method for studying the function of genes and proteins in vivo and in vitro, and is to create knock-in non-human animals by introducing genes involved in human diseases into non-human animals. Non-human transgenic animals are created by genetic manipulation such as creating knockout non-human animals by knocking out homologous genes in non-human animals of genes involved in human diseases, and these animals are used as human disease model animals. It is widely used. In particular, mice are animals to which the gene knockout / knockin technique is easily applied, and are widely used as human disease model mice.

  However, due to anatomical differences, differences in susceptibility to diseases, differences in cognitive function, etc. between rodents and primates, the results obtained using human disease model mice are directly applied to humans. Things can be difficult. Therefore, by using primates close to humans as human disease model animals, research on regenerative medicine for nerves and organs and research on higher-order functions of the brain, which are involved in common mechanisms in primates, are more accurate. It is expected to make a high effectiveness assessment. In the production of model mice, it is a common practice to return to the mother's womb regardless of the success or failure of gene transfer, and to check whether gene transfer has been performed for a large number of offspring obtained. In addition, in model animals of primates, the efficiency of introducing foreign genes into early embryos is low, and from the ethical point of view, it is possible to breed a few animals for a long period of time to obtain maximum information collection and improve the welfare of animals themselves. And relief and elimination of pain are required. In addition, in the production of such a transgenic animal, it is necessary to determine whether or not a gene has been introduced within several days when the fertilized egg reaches the blastocyst, and long-term culture is carried out as in the in vitro cell experiment. Its use as a marker for antibiotic resistance genes that are required is considered to be undesirable.

  So far, as a method for introducing foreign genes into the genomic DNA of primates, there is a method similar to rodents, such as a method of microinjecting a DNA vector for fertilized egg injection such as a DNA fragment for fertilized egg injection, a micropipette injected into fertilized eggs Inject the target foreign gene into the unfertilized ova together with the spermatozoa under the microscope using ICSI-Tr (intracytoplasmic sperm injection mediated transgenesis) method or the initial primate embryo is treated with sucrose to make the volume of the egg cavity A method for producing a human disease model primate by introducing a lentiviral vector containing a human foreign gene operably linked to a promoter into the periplasmic space of an early embryo and introducing the foreign gene (eg, a patent) Reference 1) has been proposed, but in the case of microinjection or ICSI-Tr method, its success rate And it is said to about 5%, the addition, in the case of using the lentiviral vectors as viral vectors, its success rate is that only about 20%. In addition, such lentiviral vector method is considered to be difficult to introduce a foreign gene of 8.5 kb or more, and development of a method capable of inserting a larger gene is desired.

  On the other hand, the transposon is B.I. It is a gene that moves on a chromosome that was first revealed by macrintok, and for this transposon, a control gene that changes the expression of the anthocyanin gene contained in corn seeds and plants, the gene of yellow starch in endosperm, etc. It is known that one chromosome moves to another chromosome.

  Among them, piggyBac, which is a transposon derived from lepidopteran insect virus, is known to be usable in cells derived from human, mouse and rat without increasing the risk of developing a tumor, and stable to silkworm chromosomes using piggyBac transposon Methods for introducing and expressing the protein encoded by the foreign gene have been studied using jellyfish green fluorescent protein as a model, and it has also been confirmed that the gene is stably transmitted to the offspring by mating (for example, see Non-patent Document 1) ).

  In addition, a method for producing a transgenic non-human vertebrate containing a genomic DNA of a cell containing a piggyBac-like transposon having an insert of at least 1.5 kb has been proposed (see, for example, Patent Document 2). In the study of piggyBac transfer, 34.8% (62/184) of founders were PB in analysis of transfer in transgenic mice when pronuclear co-injection of transposon donor plasmid and transposase helper plasmid was performed. [Act-RFP] single positive, 0.5% (1/184) positive for Act-PBase, 2.7% (5/184) double positive And it is far from obtaining 100% of transgenic animals. That.

International Publication WO2009 / 096101 Pamphlet JP, 2008-545375, A

Nature Biotechnology 18, 81-84, 2000

  It is an object of the present invention to introduce a gene larger than before into non-human animal cells in order to create a transgenic non-human model animal of a gene associated with higher function, and a transgenic animal The purpose is to provide a means for introducing a gene whose acquisition rate is close to 100%.

  As described above, conventionally, the probability of obtaining a target transgenic animal using a transposon was very low. As one of the reasons, for example, when a plasmid containing a transposon is injected into a cell for the purpose of introducing a genomic DNA encoding a fluorescent protein, the intended fluorescent protein is introduced into the genomic DNA of a host cell. Even if the coding DNA is not introduced into the genomic DNA, for a while after injection, the fluorescent protein fluoresces in the cytoplasm or nucleus so that it is possible to determine the cells into which the DNA encoding the fluorescent protein has been introduced genomic DNA. It is necessary to continue culture and growth of the cells for a considerable period of time, and when the cells are fertilized eggs, the cells in which the DNA encoding the fluorescent protein has been introduced into the genomic DNA at an appropriate time are temporary parents Porting was difficult.

For example, when using the conventional piggyBac Transposon Vector System, it may be possible to obtain a stable strain in which GFP continues to be expressed as a result of long-term culture , but the puromycin drug Although GFP-positive strains are obtained by selection, even if GFP-positive cells are selected by sorting, it is expected that GFP-negative cells will be produced among them unless puromycin is applied. However, if it becomes possible to select transgenic cells in a shorter period of time, it is possible to see the difference between the expression before (episomal expression) and the later expression of gene transfer to chromosomes, and for cells and embryos that undergo short-term culture. It is particularly effective for gene transfer screening.

  The present inventors have introduced a 5 'end piggyBac transposon sequence including an inverted terminal sequence (ITR) (inverted terminal repeats) to introduce DNA encoding a foreign protein into host genomic DNA. A foreign protein promoter DNA that functions in mammalian cells and a foreign protein operably linked to the downstream of the promoter DNA are encoded between the 3 'end piggyBac transposon sequence that includes the 3' inverted tail sequence. A piggyBac transposon vector operably linked to a transposon region containing the DNA to be detected, a DNA encoding a fluorescent protein for determination downstream of the promoter DNA for determination which functions in mammalian cells and upstream of the ITR sequence, and piggyBac transposase We examined using . When a transposon vector using GFP ((Green Fluorescent Protein) as the above foreign protein and KO (Kusabira-Orange: Kusabira orange) as the fluorescent protein for determination was injected into human 293T cells together with the piggyBac transposase, In cells in which the fluorescence of the fluorescent protein for determination was rapidly attenuated by the day, it was found that the DNA encoding the GFP was introduced into the genomic DNA of the cells.

  Similar examinations were carried out on mouse fertilized eggs and primate fertilized eggs. After injection of the piggyBac transposon vector, the above-mentioned KO fluorescence was rapidly attenuated by 24 hours, and in cells in which GFP was continuously expressed, It was confirmed that the DNA encoding the foreign protein GFP was introduced into the genomic DNA. In addition, in the production of a transgenic non-human model animal, GFP fluorescence is rapidly attenuated and GFP is encoded by selecting an embryo expressing GFP and transferring it to a temporary parent 96 to 120 hours after transposon injection. The success rate of the creation of transgenic non-human model animals whose genes have been integrated into the genome of fertilized egg cells has dramatically increased. Until now, selection of cells into which a foreign gene has been introduced is not impossible, but its probability is low, and often relied on the intuition and know-how of the experimenter. It has been confirmed that the probability that cells in which the fluorescence of the fluorescent protein has been rapidly attenuated can be selected as cells into which foreign genes have been introduced is significantly increased, and the present invention has been completed.

That is, the present invention is specified by the matters described in the following claims.
[1] A vector for introducing a foreign gene into a mammalian cell, comprising (A) a foreign gene transfer determining region and (B) a piggyBac transposon region;
The (A) foreign gene introduction determination region codes (a1) a promoter DNA for determination that functions in mammalian cells, and (a2) a fluorescent protein for determination operably linked downstream of the promoter DNA for determination. (B1) a pair of piggyBac transposon sequences recognized by the piggyBac transposase, wherein the (B1) piggyBac transposon region is added to the 5 'end and the 3' end of the And (b3) a DNA encoding a foreign protein comprising a fluorescently labeled protein operably linked to the downstream of the foreign protein promoter DNA, and (b3) A vector for foreign gene transfer characterized by
[2] The vector according to the above-mentioned [1], wherein the foreign protein containing a fluorescently labeled protein further contains one or more target proteins in addition to the fluorescently labeled protein.
[3] The vector according to [1] or [2] above, wherein the foreign gene transfer determination region further comprises (a3) a DNA encoding a protein destabilizing peptide.
[4] The vector according to any one of the above [1] to [3], wherein the fluorescence emitted from the fluorescent protein in the foreign gene transfer determination region and the fluorescence emitted from the fluorescently labeled protein are in a complementary color relationship.
[5] A vector kit for foreign gene transfer, comprising the piggyBac foreign gene transfer vector according to any one of the above [1] to [4], and piggyBac transposase.
[6] A method for selecting a foreign transgenic cell comprising the following steps (i) to (iv):
(A) preparing the foreign gene transfer vector according to any one of the above [1] to [4];
(B) preparing mammalian cells;
(Iii) injecting the foreign gene transfer vector prepared in (ii) above and piggyBac transposase into cells;
(D) selecting, as a foreign gene-transduced cell into which a foreign gene has been introduced, a cell in which the fluorescence of the fluorescent protein for determination by the vector for foreign gene introduction is rapidly attenuated;
[7] The method of the above-mentioned [6], wherein the foreign gene-transduced cell is a fertilized egg.
[8] A non-human mammalian transgenic animal comprising a step of transplanting a foreign gene-introduced fertilized egg selected by the method of the above-mentioned [7] onto a non-human mammalian temporary mother body and obtaining an offspring. How to make it.
[9] A nonhuman mammalian transgenic animal cell having a foreign gene and having fertility and fertility, which is obtained from the nonhuman mammalian transgenic animal produced by the method of the above-mentioned [8].

FIG. 2 is a schematic view of a vector containing a foreign gene transfer determination region and a piggyBac transposon region. (A) shows the structure of CMV-h (humanized: human) KO1-d2PEST-polyA-PB5'TR-CMV promoter-eGFP-polyA-PB3'TR, and (b) shows the CMV promoter-hKO1- It is a structure of d2PEST-polyA-CMV promoter-hKO1d2PEST-polyA-PB5'TR-CMV promoter-eGFP-polyA-PB3'TR. It is the figure which observed the expression of EGFP and KO sequentially about 293T cells after transfecting a KO / EGFP-PB transposon containing cyclic vector and a piggyBac transposase containing cyclic vector. The observation by the inverted fluorescence microscope is shown in (a1) and (b1). FACS analysis is shown in (a2) and (b2). It is the figure observed by the inverted fluorescence microscope at the time of carrying out microinjection to the male pronucleus of mouse embryo. When (a) is microinjected with a KO / EGFP-PB transposon-containing linear vector and with piggyBac transposase mRNA, (b) is when microinjected with a KO / EGFP-PB transposon-containing linear vector: (C) is the figure which observed the cell presumed that EGFP was inserted in genomic DNA. It is the figure observed by the inverted fluorescence microscope at the time of microinjection of (a) linear vector or (b) circular vector containing DNA which codes EGFP. The results of RT-PCR of the blood of wild type (negative control), the placenta of transgenic marmoset pups 1 and 2 and the hair roots of pups 1 and 2 and the skins of pups 1 and 2 and pups 1 and 2 are shown. FIG. 5 shows the construction of a mutated LRRK2-containing piggyBac vector. It is the figure which observed the expression of EGFP and KO sequentially about 293T cells after cotransfecting with a mutant LRRK2 containing piggyBac vector and a piggyBac transposase circular vector. FIG. 6 shows the construction of the piggyBac vector containing the G protein-coupled receptor 56 (gpr56) minimal promoter. FIG. 7 shows the time course of observation of Cerulean, EGFP and KO expression and the light emission of the mouse neurosphere after cotransfecting the gpr56 minimal promoter-containing piggyBac vector with the piggyBac transposase vector. It is a figure which microinjected gpr56 minimal promoter containing piggyBac vector and piggyBac transposase mRNA into the male pronucleus of mouse embryo, and observed fluorescence expression with an inverted fluorescence microscope temporally.

  The vector for foreign gene transfer into a mammalian cell of the present invention is a vector comprising (A) a foreign gene transfer determination region and (B) piggyBac transposon region, and comprising piggyBac transposase (transposon transfer enzyme) A vector capable of incorporating foreign gene DNA into mammalian cell (host cell) genome by using a known piggyBac transposon transfer mechanism by using, wherein the (A) foreign gene transfer determination region is (A1) a promoter DNA for determination which functions in mammalian cells, and (a2) a DNA encoding a fluorescent protein for determination operably linked downstream of the promoter DNA for determination, which is the above (B) piggyBac Transposon regions are (b1) 5 'end side and 3 A pair of piggyBac transposon sequences recognized by the piggyBac transposase added terminally, and (b2) a foreign protein promoter DNA functional in mammalian cells, which is disposed between the transposon sequences, and (b3) the foreign protein The region is not particularly limited as long as it is a region characterized by containing a DNA encoding a foreign protein including a fluorescently labeled protein operably linked downstream of the promoter DNA, and the (B) piggyBac transposon region is A) It can be located upstream or downstream of the foreign gene transfer determination region. The piggyBac foreign gene transfer vector of the present invention can also be used as a foreign gene transfer vector kit comprising the piggyBac foreign gene transfer vector and piggyBac transposase.

  According to the piggyBac transposon transfer mechanism, the piggyBac transposase recognizes a pair of transposon sequences present in the transposon region of (B) above, and cuts out the region containing the foreign gene DNA sequence in the transposon sequence TTAA sequence, The foreign gene can be introduced into the genomic DNA of the host cell by inserting the DNA of the excised foreign gene into the TTAA sequence region in the genomic DNA of the non-human mammalian (host) cell.

  In the present invention, examples of mammalian cells include mice, rats, rabbits, cats, dogs, pigs, cattle, etc., as well as non-human primates such as monkeys and marmosets, and human cells.

  In addition, in the case of using the kit of the present invention for the purpose of producing a non-human primate model animal, in terms of cost and ethically strongly requiring prevention of euthanasia, Are primates belonging to the gibbade family such as gibbons, rhesus macaques (Macaca mulatta), primates belonging to the cynomologous monkeys (Macaca fascicularis), common marmosets (Callithrix jacchus), pygmy marmosets (Cebuella pygmaea), cotton pistols (Satuinus) Primates such as primates belonging to the marmoset family such as oedipus), acatetamarin (Sauinaus midas), golden lion tamarins (Leontopithecus rosalia), primates belonging to the Loris family such as slow loris (Nycticebus coucang) etc. , Sexually mature at one and a half years of age, can obtain 2 to 3 litters per delivery Common marmosets are particularly preferred in that they are small in size and easy to breed because they have an average gestation period of 150 days and can be pregnant even during lactation and thus can deliver twice a year. preferable.

  The mammalian cell (host cell) in the present invention is not particularly limited as long as it is a cell capable of achieving the effects of the present invention, but a fertilized egg which is a single cell; Two-cell stage embryos, four-cell stage embryos, eight-cell stage embryos, morula embryos and / or early embryos such as blastocyst stages; pluripotent stem cells can be preferably mentioned, but development of mosaic embryos Fertilized eggs and pluripotent stem cells of one cell are preferred in that they can prevent and create excellent disease model animals for drug mechanism research and disease mechanisms generated from a single embryo.

  As a fertilized egg which is one cell described above, one cell which can be grown by somatic cell division can be mentioned, which is formed by fertilization in which a sperm enters the ovum and the nucleus of the sperm and the nucleus of the egg fuse. In detail, there can be mentioned fertilized eggs by internal fertilization collected (collected eggs) after fertilization in vivo, fertilized eggs by in vitro fertilization, and fertilized eggs obtained by freezing and storing these fertilized eggs and then thawing them.

  The pluripotent stem cells include embryonic stem cells (ES cells) isolated from early embryos and embryonic germ cells (EG cells) isolated from primordial germ cells in fetal stage (EG cells) See, eg, Proc Natl Acad Sci US A. 1998, 95: 13726-31) and germline stem cells (GS cells) isolated from the postnatal testis (eg Nature. 2008, 456: 344-). 9) and induce differentiation of somatic cells by introducing a plurality of genes into somatic cells such as bone marrow-derived stem cells, adipose tissue-derived stem cells and so on, and skin cells and so on. There can be mentioned induced pluripotent stem cells (iPS cells) having the same pluripotency as ES cells, and usually, the Oct3 / 4 gene, Klf4 gene, c-Myc and Sox2 gene are introduced. IPS cells obtained by At Biotechnol 2008; 26: 101-106) can be exemplified, but it is also known that, in the case of marmosets, Nanog and Lin 28 are required.

  Examples of the piggyBac transposon include transposons derived from nuclear polyhedrosis virus that infects Trichopulsia ni belonging to Lepidopteran, and may further include modified products thereof.

The pair of transposon (TR) sequences is not particularly limited as long as it is a pair of ITRs present on the 5 'end side and the 3' end side recognized by the piggyBac transposase having a TTAA sequence at both ends, As a general formula of the transposon sequence of 5 ', the transposon sequence at the 5' end side is a complementary double-stranded 5'-TTAAX ₁ X ₂ . . . . . . X _n-1 X _n -3 '
3'-AATTY ₁ Y ₂ . . . . . . When Y _n-1 Y _n -5 ',
3 'transposon sequence at the end side, complementary double-stranded _{5'-Y m} Y _m-1 consisting upstream of m of the nucleotide sequence of the following TTAA. . . . . . Y ₂ Y ₁ TTAA-3 '
_{3'-X m X m-1} . . . . . . X ₂ X ₁ AATT-5 '
(However, n or m bases can be present with an appropriate number of arbitrary bases, and n ≠ m or n = m)
Can be shown as

  As a specific transposon sequence, there is a piggyBac transposon sequence (5'TR) at the 5 'end side consisting of 313 bases shown in SEQ ID NO: 1 or a piggyBac transposon at the 3' end side consisting of 235 bases shown in SEQ ID NO: 2 The sequence (3'TR) can be exemplified. The details of each TR sequence are: ITR of 13 nucleotides CCC TAGAAAGATA (SEQ ID NO: 3) located downstream of TTAA of 5 'TR and 19 nucleotides TGCGTAAAATTGACGCATG (SEQ ID NO: 4) further downstream of 3 bases A sequence containing an ITR, an ITR of a 13-base TACTTTTCTAGGG (SEQ ID NO: 5) located upstream of the TTAA in the 3'TR, and an ITR of a 19-base CATGCGTCAATTTTACGCA (SEQ ID NO: 6) located further upstream of the 31 bases The sequence can be exemplified, and it is a sequence that exerts the gene transfer action of the transposon by the piggyBac transposon and the piggyBac transposase that the target gene can be cut and pasted when it is sandwiched between the 5'TR and the 3'TR. To the extent, may be one or more bases of the exemplified pair of transposon sequences include deletion, substitution or addition of nucleotide sequence.

  The promoter DNA for (a1) determination in the above (A) foreign gene transfer determination region is not particularly limited as long as it is a promoter DNA that functions in mammalian cells, and CAG (chicken β-actin) promoter, PGK (phosphoglycerate) Kinase) promoter, promoter derived from mammalian cells such as EF1α (elongation factor 1α) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, retrovirus promoter, polyoma virus promoter, adeno Although viral promoters such as viral promoters, EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers), etc. can be exemplified, CAG promoter and CMV promoter are preferable. I'm sorry.

  The DNA encoding the (a2) determination fluorescent protein in the (A) foreign gene introduction determination region may be a DNA encoding the fluorescent protein operably linked downstream of the promoter DNA of (a1) above. There is no particular limitation, and as the above-mentioned fluorescent protein, GFP that emits green fluorescence by irradiating excitation light of around 395 nm and EGFP that emits high-sensitivity green fluorescent protein by irradiating excitation light of around 480 to 500 nm Enhanced Green Fluorescent Protein) YFP that emits yellow fluorescence by emitting excitation light at around 500-530 nm, Venus that is a mutant of YFP, and orange fluorescence by emitting excitation light at around 540-560 nm KO such as KO1 and KO2 to emit, excitation light around 430 to 440 nm For example, CFP (Cyan Fluorescent Protein) such as Cerulean emitting cyan fluorescent light can be exemplified by irradiating a light, but it can be detected for a long time when the gene is expressed and does not inhibit embryo development. Fluorescent proteins such as Cerulean, GFP, EGFP, KO and the like are preferred. Luminescent proteins such as luciferase can also be included here as fluorescent proteins for convenience. The present inventors have confirmed that hKO (humanized Kusabira-Orange) sufficiently exerts an effect as KO also in marmosets and mice.

  In the present invention, "operably linked" means that when the sequences of each DNA are linked in a row and the DNA encodes a protein, the reading frame of each is matched. The 3 'end and the 5' end of the downstream DNA may or may not be directly linked as long as the effects of the present invention can be obtained, but preferably they are directly linked. In addition, it is possible to further link a poly A region (pA) that is considered to have the effect of promoting mRNA translation and stability, downstream of the DNA encoding the above-mentioned determination fluorescent protein.

  The foreign gene transfer determination region of (A) may further comprise (a3) a DNA encoding a protein destabilizing peptide operably linked downstream of the promoter DNA of (a1). By the action of the protein destabilizing peptide, the above-mentioned fluorescent protein for determination can be degraded faster.

  The protein destabilizing peptide in the present invention is not particularly limited as long as it is a peptide having an action of shortening the half life of such protein in vivo by being fused to a target protein, and such half life is shortened The action is to raise the sensitivity of the fusion protein containing the target protein to peptidases, and the action to shorten the half life of the target protein as the target protein is more rapidly degraded by the peptidase. Can. Such a protein destabilizing peptide can be fused on either the N-terminal side or C-terminal side of the DNA encoding the determination fluorescent protein, and can also be fused via a linker sequence or the like.

  As the amino acid sequence of the above-mentioned protein destabilizing peptide, P; proline, E; glutamic acid, S; serine, T; PEST sequence containing a large amount of threonine, C-terminal of short-lived proteins such as uracil permease A region-derived sequence, a CL1 sequence can be mentioned. Specifically, the d2PEST sequence shown in SEQ ID NO: 7, the d4PEST sequence shown in SEQ ID NO: 8, and one or more amino acids of these peptide sequences are substituted, added, deleted or inserted, and the peptide and the substance It is possible to exemplify peptide sequences having identical protein destabilizing activity. Here, "dn" of the dnPEST sequence indicates the proteolysis time, and indicates that the smaller the value of n, the faster the degradation. In addition, commercially available products such as destabilized fluorescent protein (manufactured by Clontech) constructed by fusing a part of mouse ornithine decarboxylase (MODC) in which a PEST domain is present at the C-terminus of the fluorescent protein can also be used.

  The (b2) foreign protein promoter DNA disposed between the pair of transposon sequences is not particularly limited as long as it is a promoter DNA sequence that functions in mammalian cells, and includes the promoters exemplified as the aforementioned promoter DNA for determination. It may be either a DNA having the same sequence as that of the DNA for the determination promoter or a DNA having a different sequence, as long as expression of any protein is not significantly reduced by promoter interference.

  The DNA encoding a foreign protein (b3) disposed between the pair of transposon sequences is a DNA encoding a foreign protein including a fluorescently labeled protein operably linked downstream of the foreign protein promoter DNA The foreign protein is not particularly limited, and in addition to the fluorescently labeled protein, a target protein capable of changing the host's intact function can be mentioned. The foreign protein is, for example, 1) fluorescently labeled protein 2) A configuration including a fluorescently labeled protein and one or more target proteins, 3) one or more target proteins, etc. can be exemplified.

  The gene for the target protein is a protein other than the above-mentioned fluorescent protein and is a causative gene for Parkinson's disease, a mutant gene of mutant alpha synuclein gene, a mutant gene of LRRK2 (Homo sapiens leucine rich repeat kinase 2), a gene for CFC syndrome Overexpression of genes such as mutant genes of BRAF, mutant genes of DNA encoding Ca-independent group 6 phospholipase A2 (Pla2g6) considered to be a cause of neurogenic muscular atrophy Examples include Huntingtin gene which is a causative gene of Huntington's disease, HNF1αP291fsinsC which is a causative gene of juvenile adult onset diabetes, regucalcin protein and the like.

  As the above-mentioned LRRK2 gene, a gene that can be represented by the 9239 bp base sequence of Homo sapiens leucine rich repeat kinase 2 mRNA shown in NCBI Reference Sequence: NM_198578.3 can be exemplified.

  The mutation gene of LRRK2 is a cause of Parkinson's disease in which LRRK2 is inactivated by deleting at least a part of the nucleotide sequence of DNA encoding LRRK2 and inserting or substituting another nucleotide sequence with another nucleotide sequence. And a mutant gene (SEQ ID NO: 9) encoding a mutant protein in which a glycine residue at position 2019 of LRRK2 is substituted with a serine residue.

  A Parkinson's disease model non-human mammal can be produced by introducing such a mutant gene into genomic DNA of a host non-human mammal.

  Examples of the above-mentioned foreign protein promoter DNA include the promoters exemplified in the above-mentioned promoter DNA for determination. In addition, when producing a non-human mammal, a DNA encoding a fluorescently-labeled protein operably linked downstream of a promoter DNA 1 that functions in embryos but does not function in embryos, such as the aforementioned EOS (first expression When the transposon region comprises a cassette) and a foreign gene DNA (second expression cassette) containing a target protein operably linked downstream of an adult-functioning promoter DNA 2, the fluorescently-labeled protein is expressed in the embryo These cells are superior in that they can produce non-human mammals that do not express fluorescently labeled proteins in adults.

  The fluorescently labeled protein can be selected from the fluorescent proteins exemplified in the above-mentioned fluorescent protein for determination, but in order for the determination in the fluorescent protein for determination to be performed efficiently and rapidly, visual observation or FACS etc. In order to be able to distinguish between the fluorescently labeled protein and the fluorescent protein for determination by means of the above, it is necessary that the fluorescent labeled protein emits a wavelength different from the wavelength of the fluorescence emitted by the fluorescent protein for determination. When a fluorescent protein having a color close to or related to the color of the fluorescent protein for the color or a complementary color is selected as the fluorescent labeled protein and used as a preferable combination of the fluorescent labeled protein and the fluorescent protein for determination, the fluorescent labeled more easily. Visually distinguish between protein and the above-mentioned fluorescent protein for determination Preferable in that it is Rukoto.

  Specific combinations of the above-mentioned fluorescently labeled protein and the above-mentioned fluorescent protein for determination include: green fluorescent EGFP and orange fluorescent orange, and green fluorescent GFP and orange fluorescent Orange-fluorescent orange-fluorescent orange and green-fluorescent EGFP, orange-fluorescent green-flavored orange-GFP and green-fluorescent GFP, yellow-fluorescent YFP and cyan-fluorescent CFP, cyan-fluorescent A combination of CFP and YFP that emits yellow fluorescence, CFP that emits cyan fluorescent light and Caffira orange that emits orange fluorescent light, or a combination of Caffira orange that exhibits orange fluorescence and CFP that emits cyan fluorescent light, and fluorescent proteins of these And each combination such as luciferase and luciferase It can Shimesuru.

Examples of the configuration of the foreign gene-containing vector in the present invention include a configuration including CMV-hKO1d2PEST-polyA-PB5'TR-CMV promoter-EGFP-polyA-PB3'TR (FIG. 1 (a)). There is no limitation as long as it exerts an effect in the present invention, for example, CMV promoter-hKO1-d2PEST-polyA-CMV promoter-hKO1-d2PEST-polyA-PB5'TR-CMV promoter-EGFP-polyA- PB3'TR (FIG. 1 (b)), CMV-hKO1d2PEST-PB5'TR-CMV promoter-EGFP-PB3'TR, CMV-hKO1d2PEST-CMV-hKO1d2PEST-PB5'TR-CMV-EGFP-PB3'TR, CMV ₁ promoter -hKO D2PEST- poly A-PB5'TR-CAG promoter - and DNA-2A-EGFP- poly A-PB3'TR encoding a target protein, CMV ₁ promoter -Cerulean-d2PEST- poly A-PB5'TR-hgpr56-eGFP- Mention may be made of the vector comprising the luciferase-Ins-EOS-hKO1-polyA-PB3'TR.

  In the present invention, the piggyBac transposase includes, in addition to the piggyBac transposase (protein) itself, a cyclic or linear vector containing a DNA sequence encoding the piggyBac transposase, and a DNA encoding the piggyBac transposase PiggyBac transposase expression products such as transcribed mRNA (piggyBac mRNA) and cDNA can be included for convenience, but it can be expected that expression is faster, and it is not preferable that transposase itself is integrated into the genome. PiggyBac transposase mRNA can be suitably mentioned in that it is desired to stop only by transient expression.

  As a method of preparing the above piggyBac transposase expression product, there is a method of preparing a circular vector containing DNA encoding piggyBac transposase by a conventionally known method, or such circular vector is linearized using an appropriate restriction enzyme. The method of preparing the vector, the method of preparing mRNA including the coding region of piggyBac transposase by T7 RNA polymerase using such a circular vector or linear vector as a template, or recombinant expression by expressing as a protein by E. coli etc. The method of preparing the piggyBac transposase can be exemplified.

  The mode of foreign gene transfer into the genomic DNA of mammalian cells using the transposon in the present invention is a random insertion mutagenesis system, and the present invention is applied to the male pronucleus of the fertilized egg or the female pronucleus of the fertilized egg or the fertilized egg cytoplasm of the fertilized egg. By injecting a vector kit, hemizygote mutations can be created.

In the present invention, a method comprising the steps of (a) to (d) below can be exemplified as a method for selecting a foreign gene-transduced cell, and such a method comprises the vector kit for foreign gene transfer of the present invention described above: It is preferable to use
(A) preparing a vector for introducing a foreign gene;
(B) preparing mammalian cells;
(Iii) injecting the foreign gene transfer vector prepared in (ii) above and piggyBac transposase into cells;
(D) selecting a cell in which the fluorescence of the fluorescent protein for determination by the vector for foreign gene transfer has been rapidly attenuated as a cell into which the foreign gene has been introduced;
The method of selecting a foreign transgenic cell is also a method of improving the selection probability of the foreign transgenic cell.

  In the step of preparing the vector for foreign gene transfer of (i) above, as a method for producing the vector for foreign gene transfer, a conventionally known method can be used. For example, a Gateway cloning system can be used. . The Gateway cloning system is based on a site-specific recombination system involved in the entry of λ phage into the E. coli chromosome, and facilitates construction of an expression vector by using the Gateway signal (att). . An entry vector (attL1 and attL2 sequences at both ends) in which the target gene is integrated by reacting (BP reaction) between a donor vector having attP1 and attP2 sequences and attB1 and attB2 sequences added at both ends of the target gene The target gene is then inserted by recombine reaction (LR reaction) with this entry vector and the destination vector (attR1 and attR2 sequences added) in which the promoter necessary for expression is integrated. Is a method of producing a vector (expression vector). Specifically, for example, PB-CA-rtTAAdv, which is a destination vector containing a transposon region purchased from Addgene, may be used, and the promoter in PB-CA-rtTAAdv may be modified and / or The Gateway part can be modified. Moreover, it is also possible to produce the entry vector containing PB-CA-rtTAAdv, a promoter, and GFP using KO / EGFP-PB transposon containing cyclic vector supplied by Medical Research Council National Laboratory (UK). In addition, a method of using a vector that does not reduce the developmental potential of embryos is preferable.

  In the step (b) of preparing mammalian animal cells, the mammalian cells can be prepared using a method known in the art, and for non-human primates, methods of in vitro fertilization and fertilized eggs As the method of collection, methods described in JP 2012-125410 and WO 2009/096101 can be exemplified.

  In the step of injecting the foreign gene transfer vector and the transposase into the cell of (c), the foreign gene is introduced into the cell together with the piggyBac transposase vector together with the piggyBac transposase. The method is not particularly limited as long as it can be introduced into cells, for example, mRNA prepared using foreign gene-containing vector and piggyBac transposase vector or transposase vector as a template, opti-MEM (manufactured by Thermo Scientific) or physiological saline Can be lipofected with Lipofectamine; injected by microinjection; electroporation; and the like. Preferred is a method of lipofection of a yBac transposon circular vector and a piggyBac transposase circular vector using an opti-MEM solution. When injecting into a fertilized egg, the foreign gene-containing piggyBac transposon circular vector and transposase mRNA And a method of microinjection using physiological saline, and when injecting into a neurosphere, a method of electroporating a foreign gene-containing piggyBac transposon circular vector and transposase mRNA is preferable. It can be mentioned.

  The site of the cell into which the foreign gene-containing vector is injected together with the transposase is not particularly limited as long as it can exert the effects of the present invention, but to minimize the development of mosaic embryos Injection into the male pronucleus, female pronucleus, cytoplasm, etc. of the fertilized egg at cell stage is preferable, and in the case of microinjection, it is easy to inject because the male pronucleus is larger than the female pronuclei. In point, male pronuclear injection can be mentioned suitably. A male pronucleus is a sperm-derived nucleus formed by a fertilized egg, which is formed at the end of the fertilized egg, then moves to the center of the fertilized egg, fuses with a female pronucleus, and A nucleus is made. The injection amount of the solution may be such that it satisfies the volume of the injection site in the fertilized egg, for example, when the injection site in the fertilized egg of marmoset is a male pronucleus, 1 to 10 pL is preferable, 2 to 2 8 pL is more preferable, 3 to 7 pL is more preferable, and 4 to 6 pL is particularly preferable. In addition, even when injected into the cytoplasm by microinjection, since it is possible to introduce foreign genes into genomic DNA by the action of transposase, cytoplasmic injection is possible for primates such as marmosets, which have small pronuclei and are difficult to inject. It is valid.

  The step of selecting a cell into which the foreign gene of (d) has been introduced is not particularly limited as long as it is a step of selecting a cell in which the fluorescence of the fluorescent protein for determination is rapidly attenuated. For cells in which the fluorescence is rapidly attenuated, the transposon recognizes and cuts out the transposon sequence of the foreign gene-containing vector injected into the cell, thereby inserting the transposon region into the host cell genomic DNA. The fluorescence intensity due to the expression of the DNA encoding the determination fluorescent protein in the foreign gene introduction determination region which has not been inserted is significantly smaller than the fluorescence intensity of the determination fluorescent protein in the vector which has not been excised by transposase. Can be mentioned. Specifically, in the case of human 293T cells, when the transposon region is not introduced into the host cell, the fluorescent protein for determination is expressed until the fifth day after vector injection, and is gradually attenuated by the fifteenth day On the other hand, when introduced into the host cell, the fluorescence of the fluorescent protein for determination almost disappears by the fifth day after vector injection. In addition, in the case of mouse fertilized eggs, when the transposon region is not introduced into the host cell, the fluorescent protein for determination is expressed from the fourth day to the fifth day until the blastocyst stage after vector injection, When introduced into a host cell, the fluorescence of the fluorescent protein for determination can not be visually confirmed using a fluorescence microscope by the first day (24 hours) after vector injection. In the case of a primate fertilized egg, the fluorescent protein for determination is expressed until the 10th day until the blastocyst stage after vector injection when not introduced into the host cell, and when introduced into the genome of the host cell By the first day (24 hours) after vector injection, the fluorescence of the fluorescent protein for determination can not be visually confirmed using a fluorescence microscope. Therefore, when a foreign gene is introduced, the fluorescence of the fluorescent protein for determination disappears early, such as 72 hours or more, preferably 48 hours or more, more preferably 36 hours or more, still more preferably 24 hours or more after vector injection. These cells can be determined to be cells into which foreign gene transfer has been performed. However, the mechanism for accelerating the decay rate of the fluorescence of such a determination fluorescent protein has not been clarified yet.

  In the step of selecting the cell into which the foreign gene has been introduced in (d) above, when the foreign gene contains the above-mentioned fluorescently labeled protein, when the foreign gene is introduced into the host cell, cell proliferation in the host cell The fluorescence of the fluorescent protein for determination is rapidly attenuated while the fluorescence of the fluorescently labeled protein is gradually intensified. On the other hand, when the foreign gene is not introduced into the host cell, the fluorescent protein for determination and the fluorescently labeled protein maintain fluorescence for a while, but the two types of fluorescence do not increase but gradually attenuate and disappear eventually. Therefore, by selecting cells in which the fluorescence of the fluorescent protein for determination is rapidly attenuated, it is possible to use the kit of the present invention in which the foreign gene for the fluorescent protein for determination of the present invention is used in combination. The selection rate of the cells into which the foreign gene has been introduced is significantly higher when the gene is introduced.

  As a specific determination method of the said fluorescence intensity, it can carry out by visual observation by a fluorescence microscope or flow cytometry analysis by FACS. In the case of visual observation, it can be judged that fluorescence is visually recognized when observed with an inverted fluorescence microscope under, for example, a sensitivity of 400 exposure time, 1 second, or a condition having an equivalent effect thereto.

  The method for producing the non-human mammalian transgenic animals is not particularly limited as long as the collected fertilized eggs are returned to the uterus and the offspring are obtained, and it is determined that a foreign gene has been introduced into genomic DNA in the case of mice. Are transferred to the fallopian tube and uterus of a temporary parent mouse such as a pseudopregnant mouse as a non-human mammalian animal temporary mother, and cesarean section or spontaneous delivery is performed just before delivery to obtain a neonatal mouse The method can be exemplified to obtain a transgenic mouse. In the case of non-human primates, for example, the fertilized eggs are returned to the body by the method described in JP 2012-125410 or WO 2009/096101 to obtain transgenic non-human primates such as transgenic marmosets. be able to.

  In the production of transgenic marmosets, after microinjection of a PB transposon-containing cyclic vector and piggyBac transposase into the cytoplasm of marmoset fertilized eggs 15 to 2 hours after in vitro fertilization, 4-5 days (96 to 120 hours after microinjection) ) Of the embryos that developed from the 6th cell stage to the 16th cell stage, the fluorescence of the fluorescent protein in the determination region is rapidly attenuated (eg, by 24 hours after microinjection), and the expression of the fluorescently labeled protein is stable It is preferred to transplant the cells to a temporary parent.

  Whether or not a foreign gene has been introduced into the non-human primate transgenic animal cells can be confirmed, for example, by PCR using a primer set corresponding to the DNA encoding each foreign protein.

  By performing a progeny assay on a transgenic animal into which a foreign gene has been introduced, a non-human mammalian transgenic animal in which expression of the foreign protein has been confirmed can be obtained, and from such an animal a non-human having reproductive ability and / or fertility. Human mammalian transgenic animal cells can be obtained.

  The foreign gene transfer cells selected by the method for selecting a foreign gene transfer cell can also be used as a screening cell for screening an agent for ameliorating a disease caused by the transferred foreign gene.

  For example, a foreign transgenic cell into which a mutant gene of LRRK2 has been introduced as a foreign gene can be used as a cell for screening a therapeutic agent for Parkinson's disease. For example, a mutation in which the glycine residue at position 2019 of LRRK2 is a serine residue has been shown to cause Parkinson's disease by causing excessive serine / threonine phosphorylation activity of LRRK2 In neural stem cells carrying the LRRK2 mutation, depending on passage, various substrates are hyperphosphorylated, and it is believed that they may be responsible for phenotypes such as nuclear membrane deformation. Therefore, a neural stem cell having the above LRRK2 mutation that has been passaged repeatedly, and a cell with a phenotype such as deformation of the nuclear membrane, is rescued when deformation of the nuclear membrane is rescued by adding a test substance to the medium. The test substance can be picked up as a candidate substance for treating Parkinson's disease.

  Hereinafter, the present invention will be more specifically described by way of examples, but the technical scope of the present invention is not limited to these examples.

Example 1
[Foreign gene transfer to human embryonic kidney 293T cells]
A DNA encoding an EGFP protein is expressed as a genomic DNA of human embryonic kidney 293T cells, using a KO / EGFP-piggybac (PB) transposon-containing circular vector and a piggyBac transposase vector (Medical Research Council (UK)). I tried to introduce it. The human embryonic kidney 293T cells are cells in which SV40T has been introduced into 293 cells which are human fetal-derived kidney epithelial cells.

(Preparation of human embryonic kidney 293 T cells)
DMEM medium (Dulbecco's modified Eagle's medium) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic solution of human embryonic kidney 293T cells (Riken BioResource Center) It was grown in.

[Preparation of KO / EGFP-PB Transposon-Containing Circular Vector]
(Preparation of foreign gene transfer determination region)
The CMV ₁ promoter DNA as the determination promoter shown in SEQ ID NO: 10; the hKO 1 as the fluorescent protein operably linked downstream of the determination promoter DNA as the determination promoter DNA, is shown in SEQ ID NO: 11 DNA sequence; d2pest shown in SEQ ID NO: 7 as DNA encoding a protein destabilizing sequence acting on hKO1; CMV ₁ promoter DNA which is a promoter for determination shown in SEQ ID NO: 10 again; The DNA sequence shown in SEQ ID NO: 11 encoding hKO1 as a fluorescent protein operably linked downstream; d2pest shown in SEQ ID NO: 7 as DNA encoding a protein destabilizing sequence acting on hKO1; The co Was the foreign gene introduced determination area by sequentially connecting; DNA sequence of and a poly A shown in SEQ ID NO: 12; DNA to.

  The necessity of the above PEST sequence was preliminarily examined using 293T cells. When KO is used as a fluorescent protein for determination of the foreign gene transfer determination region and GFP is used as a fluorescently labeled protein for the piggyBac transposon region, without the PEST sequence, the KO fluorescence leaks to the GFP filter while the fluorescence is strong. I confirmed that it might be difficult. Since the expression strength of GFP differs depending on the type of promoter used, it has been confirmed that it is preferable to select KOd4PEST or KOd2PEST for each type of vector (data not shown). In the following study, it was decided to use KOd2PEST.

(Preparation of PB transposon domain)
Then, the DNA sequence of the 5 'end of the Piggybac (PB) transposon, PB5'TR (SEQ ID NO: 1), and the DNA sequence of the 3' end of the PB transposon, PB3'TR (SEQ ID NO: 2) A circular vector was constructed that contains the PB transposon region making up the repetitive sequence.

CMV ₂ promoter DNA as shown in SEQ ID NO: 13 as a foreign protein promoter DNA that functions in non-human primate cells in the PB transposon region; encoding a foreign protein operably linked downstream of the CMV ₂ promoter By using, as the DNA, a DNA encoding the EGFP gene shown in SEQ ID NO: 14; and a DNA sequence of poly A shown in SEQ ID NO: 12: PB5′TR DNA, CMV ₂ promoter DNA, DNA encoding EGFP, poly ADNA , PB3'TRDNA, as an EGFP-containing PB transposon region containing sequentially from the 5 'end.

(Constitution of a KO / EGFP-PB transposon-containing circular vector)
The destination vector obtained by modifying a promoter in PB-CA-rtTAAdv as the backbone, CMV-hKO and CMV-GFP (those riding in the entry vector) CMV ₁ promoter was inserted into the -hKO1d2PEST-CMV ₁ promoter -hKO1d2PEST-PB5 ' The specific configuration of the KO / EGFP-PB transposon-containing circular vector (SEQ ID NO: 16) containing the configuration of TR-CMV ₂ promoter-EGFP-PB3'TR is as follows.

CMV ₁ promoter DNA sequence for determination (SEQ ID NO: 10):
The nucleotide sequence of SEQ ID NO: 16 from No. 1978 to No. 2566, and from No. 3551 to No. 4139;
hKO1 (SEQ ID NO: 11):
Nucleotide sequences of SEQ ID NO: 16 from 2584 to 3237 and 4157 to 4810;
d2PEST (SEQ ID NO: 7):
The nucleotide sequences of SEQ ID NO: 16 from 3244 to 3367 and 4817 to 4940;
Poly A (SEQ ID NO: 12):
The nucleotide sequences of SEQ ID NO: 16 from 3371 to 3491 and from 4944 to 5064;
PB5'TR (SEQ ID NO: 1):
The nucleotide sequence of SEQ ID NO: 16 from 5194 to 5506;
CMV ₂ promoter DNA (SEQ ID NO: 13):
The nucleotide sequence of SEQ ID NO: 16 from 5552 to 6100;
DNA sequence encoding EGFP (SEQ ID NO: 14):
The nucleotide sequence of SEQ ID NO: 16 from 6124 to 6841;
PB3'TR (SEQ ID NO: 2):
The nucleotide sequence of SEQ ID NO: 16 from 7606 to 7840;

(Preparation of a vector for expression of piggyBac transposase)
A piggyBac transposase expression vector containing a transposase-encoding DNA sequence that recognizes a pair of inverted repeat sequences in the PB transposon region was purchased from Medical Research Council (UK). Such a vector is described in Yusa et al, PNAS 2011 as a DNA sequence encoding the above piggyBac transposase downstream of the CMV ₃ promoter DNA sequence (SEQ ID NO: 17), and the Medical Research Council National Laboratory (UK The DNA sequence (SEQ ID NO: 18) coding for HyPBase (hyperactive piggybac transposase) supplied by), and poly A (SEQ ID NO: 12) are incorporated.

(Co-transfection into 293T cells)
The KO / EGFP-PB transposon-containing circular vector and the piggyBac transposase circular vector were cotransfected into the above-mentioned 293T cells in a concentration ratio of 2: 1 in total of 1 μg / ml (Day 0). 293T cells transfected with only the KO / EGFP-PB transposon-containing circular vector served as a negative control. For the transfected 293T cells, EGFP with excitation light 488 nm, KO1 and GFP with excitation light 548-584 nm, inverted fluorescence microscope (type IX71, manufactured by Olympus; camera DP 73, sensitivity 400 exposure time 1 Observed over time. The results are shown in (a1) and (b1) of FIG. FACS analysis of EGFP and hKO1 was also performed. The results are shown in (a2) and (b2) of FIG.

(result)
As is clear from FIGS. 2 (a1) and (a2), in the negative control cells to which only the KO / EGFP-PB transposon-containing circular vector was added, expression of EGFP (green) and hKO1 (orange) was initiated on Day 1 . Also on Day 5, strong expression of both EGFP and hKO1 can be confirmed from visual observation (a1) and FACS analysis (a2) of the fluorescence of EGFP and hKO1. However, on Day 15, both of the fluorescence by EGFP and the fluorescence by hKO1 almost disappeared.

  Therefore, in the case where the piggyBac transposase vector is not added and the transposon region is not excised in the transposon-containing circular vector, coexpression of EGFP and KO continues at least until Day 5, and then by Day 15, hKO1 It was confirmed that the expression of both EGFP and EGFP disappeared. As apparent from the FACS data in FIG. 2 (a2), in the cells to which only the KO / EGFP-PB transposon-containing circular vector was added and the piggyBac transposase vector was not added, the cells into which EGFP had been introduced It was not. Since 293T cells are easily integrated at random, there may be cells into which only EGFP or hKO1 has been introduced, but it can be said that there were no cells into which EGFP had been introduced, even in consideration of the possibility.

  On the other hand, as apparent from FIGS. 2 (b1) and (b2), in the cells (b cells) co-transfected with the above-mentioned KO / EGFP-PB transposon-containing cyclic vector and the piggyBac transposase circular vector, EGFP was obtained on Day 1 The expression of KO1 and KO1 can be observed, but on Day 5, compared with the above (a) cells, the green fluorescence of GFP is strong and the intensity of orange fluorescence of KO1 is small. It was confirmed that the cells were present and that cells expressing both EGFP and KO1 were mixed. Therefore, when EGFP is introduced into genomic DNA by the action of transposase, the expression of EGFP is stabilized and enhanced, while the expression of KO1, which is not introduced into genomic DNA, is rapidly attenuated by the fifth day or so That was confirmed. On Day 15, the fluorescence of KO1 was not observed and many cells expressing only EGFP were observed, confirming that the piggyBac transposon region containing EGFP was introduced into the genomic DNA of 293T cells.

Example 2
[EGFP introduction into mouse embryo using KO / EGFP-PB transposon-containing linear vector]
We tried to insert DNA encoding EGFP protein into genomic DNA of C57BL / 6 mouse embryo. As a foreign gene-containing vector, KO / EGFP- obtained by cleaving the 412st and 8065th bases of SEQ ID NO: 16 with FspI by restriction enzyme treatment and linearizing the above-mentioned KO / EGFP-PB transposon-containing circular vector A PB transposon-containing linear vector was used. Furthermore, using the linearized piggyBac transposase expression vector as a template, piggyBac transposase mRNA was prepared by T7 RNA polymerase.

(Examination of transposase in mouse embryo)
Observation by inverted fluorescent microscope when the above KO / EGFP-PB transposon-containing linear vector and piggyBac transposase mRNA are microinjected into male pronuclei of mouse embryo using a micromanipulator (Leica) The results are shown in FIG. 3 (a). In addition, Fig. 3 (b) also shows the case where the above linear vector was microinjected but the piggyBac transposase mRNA was not microinjected. FIG. 3 (c) shows the appearance of cells after 24, 48, 72 and 96 hours after microinjection for cells in which EGFP is presumed to be inserted into genomic DNA. In addition, NC of (circle) mark in the figure is a negative control which has not performed microinjection.

(result)
As is clear from FIGS. 3 (a) and 3 (c), in the cells in which the transposon mechanism acts to insert EGFP into genomic DNA (green cells shown by the square white arrows in FIG. 3 (a)), The green fluorescence of EGFP began to be expressed after 24 hours (Day 1), whereas KO1 (orange) was hardly expressed from the beginning. It was confirmed that the development progressed to the blastocyst after 96 hours (Day 4) with the EGFP alone fluorescence maintained (FIG. 3 (c)). Therefore, when EGFP was inserted into genomic DNA, KO1 that was not inserted into genomic DNA was expected to be inactivated more rapidly than the destabilization of KO1 predicted by the action of d2pest. On the other hand, when EGFP was not inserted into genomic DNA, EGFP and KO1 were coexpressed until Day 4 (FIG. 3 (b)).

  Therefore, it could be considered that in an embryo in which KO1 was not expressed from the beginning or KO1 was not expressed by 72 hours after microinjection, the target gene was likely inserted into genomic DNA.

[Example 3]
[EGFP transfer to mouse embryo using KO / EGFP-PB transposon-containing circular vector]
Since the embryo development rate was lower than expected in the study using the above linear vector, we tried to use the transposon circular vector, which is said to have lower embryonic toxicity than the transposon-containing linear vector, to try microinjection into mouse embryos .

  We attempted to introduce EGFP-encoding DNA into mouse embryo genomic DNA. For 51 fertilized eggs derived from C57BL / 6 mice, 2 ng / μL of KO / EGFP-PB transposon-containing circular vector and 1 ng / μL of mRNA prepared from piggyBac transposase vector are dissolved in physiological saline and It was microinjected into the nucleus. In addition, about 49 C57BL / 6 mouse-derived fertilized eggs, 2 ng / μL of KO / EGFP-PB transposon-containing linear vector and 1 ng / μL of mRNA prepared from piggyBac transposase vector are dissolved in physiological saline and mouse embryo It was microinjected into the male pronucleus. The results are shown in FIG. 4 (a) (for linear vector) and (b) (for circular vector).

  As apparent from FIG. 4 (a), when a KO / EGFP-PB transposon-containing linear vector is used, the expression of green and orange fluorescence can be confirmed rapidly and strongly, and 43 embryos that expressed fluorescence are The rate was as high as 42. However, only 21 embryos expressed orange, and only 4 embryos developed to blastocyst (96 hours). As is apparent from FIG. 4 (a) at 96 h, of the four embryos that have developed to the blastocyst, there are two embryos that have fluorescence of GFP and no fluorescence of KO. It was determined that the desired GFP gene was introduced into the cell gene in As for the other two, neither GFP nor KO fluorescence was found. Red circles are negative controls (NC). On the other hand, as is clear from FIG. 4 (b), when the KO / EGFP-PB transposon-containing circular vector is used, the time when the fluorescence expression can be confirmed is delayed and the expression amount is reduced, but the blastocyst The number of embryos reached was 14 out of 41 in 96 hours, and the blastocyst rate increased. In addition, there were 31 embryos that expressed green light of EGFP and did not show expression of KO, and although the expression rate of EGFP decreased somewhat, expression of KO was not observed at all.

  From the above results, when the KO / EGFP-PB transposon-containing circular vector is used, the time when the fluorescence expression can be confirmed is delayed and the expression amount is small, but the embryonic toxicity is low and the introduction rate of EGFP into genomic DNA Was judged to be preferable to use a circular vector.

Example 4
(Preparation of transgenic mice)
A transgenic mouse was prepared by injecting the above-mentioned KO / EGFP-PB transposon-containing cyclic vector and mRNA prepared from the piggyBac transposase vector into a mouse fertilized egg by microinjection. As a fertilized egg, a B6C3 strain hybrid mouse B6C3F1 fertilized egg obtained by mating a C57BL / 6 (B6) strain mouse and a C3H strain mouse was used. The mRNA prepared from the above KO / EGFP-PB transposon-containing cyclic vector and the piggyBac transposase vector (concentration ratio 2: 1) is injected into the male pronucleus or cytoplasm of a fertilized egg for 10 hours after fertilization, and the mouse is obtained until the blastocyst The cells were cultured in an incubator at 37 ° C. in mWM medium (manufactured by Ark Resource Co., Ltd.) which is a culture medium for extracorporeal culture. Each embryo that has progressed to the blastocyst is observed with an inverted fluorescence microscope (manufactured by Olympus), and embryos in which only green fluorescence is observed are selected (color selection +) or without selection by fluorescence expression (Color selection-) A non-human mammalian animal mother was transplanted into the uterus of a pseudopregnant mouse (ICR strain), and neonatal mice were obtained by spontaneous delivery or cesarean section. Neonatal mice were raised to weaning by performing foster manipulation.

  Between 2 and 3 weeks after birth, the tail of the offspring was removed by about 5 mm to extract mouse genomic DNA. The acquisition rate of transgenic mice was investigated by PCR using each primer set shown below capable of specifically detecting EGFP or KO. The results are shown in Table 1 below.

EGFP:
5'-CAAGGACGACGCGACACTACAAGACC-3 '(forward primer) (SEQ ID NO: 19)
5'-GCTCGTCCATGCCCGAGAGTGA-3 '(reverse primer) (sequence number 20)
KO:
5'-GAGGGCCTGAGTCGGAGA-3 '(forward primer) (sequence number 21)
5'-CACCAGCCTGTGGCCGATGA-3 '(reverse primer) (sequence number 22)

(Result 1-Nuclear 1 ng / μL · With fluorescence confirmation (+))
For 92 fertilized eggs, 66 embryos (75.0) reached blastocysts (96 hours) in which 1 ng / μL of transposon vector / transposase mRNA mixture (2: 1) was microinjected into male pronuclei at 5 pL Among the%), 55 (83.3%) green fluorescence was observed visually with a microscope and no KO expression was observed. Among them, five neonatals were obtained by transferring 39 embryos into the uterus of pseudopregnant mice (ICR strain), and three weaned litters were obtained. All three of these animals expressed EGFP. However, one of them was also expressed for KO. This is considered to be the reason for random integration. From the above, when blastocyst embryos for which fluorescence by EGFP has been confirmed in blastocysts (96 hours) are transferred to the mother, the percentage of transgenic mice into which EGFP has been introduced among the obtained mice is 100%. The

(Result 2-Cytoplasm 3 ng / μL, with fluorescence confirmation (+))
For 95 fertilized eggs, 3 ng / μL of transposon vector transposase mRNA mixture (2: 1) was microinjected into the cytoplasm of male pronuclear phase. Of 64 embryos (85.3%) that reached blastocyst (96 hours), 27 showed green fluorescence by visual observation using a microscope and no expression of KO was observed (26 42.2%). Of these, 26 neonatals were obtained by transferring 26 embryos to the uterus of pseudopregnant mice (ICR strain), and 3 weanlings were born. All three mice expressed EGFP, but no expression of KO was observed. Therefore, when blastocyst embryos for which fluorescence by EGFP has been confirmed and expression of KO has not been observed, the percentage of transgenic mice into which EGFP has been introduced among the obtained mice is 100%. there were.

(Result 3-Nuclear 1 ng / μL, no fluorescence confirmation (-))
When 70 ng of fertilized eggs were microinjected with 1 ng / μL of transposon vector / transposase mRNA mixture (2: 1) into the male pronuclei, all falsely identified embryos that reached the blastocyst without any fluorescent confirmation. As a result of transplantation into the uterus of a pregnant mouse (ICR strain), the percentage of transgenic mice into which EGFP had been introduced was 50% of the obtained neonatal mice.

(Result 4-Cytoplasma 3 ng / μL, no fluorescence confirmation (-))
For 74 fertilized eggs, when microinjection of 3 ng / μL of transposon vector / transposase mRNA mixture (2: 1) into the pronuclear stage cytoplasm, fluorescence confirmation of embryos that reached blastocysts was not performed. As a result of transplantation into the uterus of all pseudopregnant mice (ICR strain), the proportion of transgenic mice into which EGFP had been introduced among the obtained neonatal mice was 81.8%.

(Summary)
As described above, the transposon vector / transposase mRNA mixture (2: 1) was microinjected into the cytoplasm of the male pronucleus or the male pronuclear stage, and the presence or absence of fluorescence was confirmed in the blastocyst, and the blastocyst was subjected to KO When expression was lost and EGFP-expressing embryos were selected, the production rate of transgenic mice into which EGFP had been introduced into genomic DNA was significantly increased, and in this study, a value of 100% was obtained. In particular, it has been shown that cytoplasmic injection of transposon vector and transposase mRNA is effective for animal species having small pronuclei and difficulty in injection.

[Example 5]
(Progeny test)
A progeny assay was performed on the transgenic mice expressing EGFP obtained above. Two mice from which embryos were injected with transposon and transposase into the male pronucleus obtained in Example 4 and in which the expression of EGFP was confirmed, and in the cytoplasm of pronuclear stage fertilized eggs, transposon and transposase mRNA Table 2 below shows the results of crossing F1 of three mice, founder B6C3 mice, with C57BL / 6J mice, which were derived from fertilized eggs into which E.sup..sup. + Was injected, and expression of EGFP was confirmed.

(result)
As shown in Table 2 above, by crossing with wild-type mice, it was possible to obtain F1 mice into which EGFP had been introduced into genomic DNA, except for the F1 strain of Nuclear 3 (injected in the male pronuclear phase). Therefore, DNA encoding EGFP introduced into cells by microinjecting mRNA prepared from the above-mentioned KO / EGFP-PB transposon-containing cyclic vector and piggyBac transposase vector is also expressed in germ cells and transmitted to the next generation It was confirmed to do.

[Example 6]
[Transgenic marmoset production]
A transgenic marmoset into which DNA encoding EGFP was introduced was produced from the common marmoset bred and maintained at the JRC.

  3 ng / μL of KO / EGFP-PB transposon-containing circular vector and piggyBac transposase mRNA (2: 1) were microinjected into the cytoplasm of marmoset fertilized eggs at 17 hours after in vitro fertilization. The cells were cultured at 37 ° C. in Blast assist medium (manufactured by ORIGIO). Observation of expression of GFP and KO was continued with an inverted fluorescence microscope (manufactured by Olympus), and the embryo developed from 6 to 16 cell stages at 4-5 days (96 to 120 hours) after microinjection. 30 mice expressing only the green fluorescence of the above were transplanted to 19 temporary parents and 3 pregnant (15.8%), one of them gave birth, 2 pups 1 and 2 (6.7%) ) Were born. On the other hand, in the embryo that developed from morula to blastocyst at 6-7 days (144-168 hours) after microinjection, 19 mice expressing only green fluorescence were transferred to 12 temporary parents. I got pregnant (33.3%) but all miscarriages. Although the reason for the abortion is not clear, it may be possible that there was damage by UV irradiation as fluorescence observation was repeated many times until it became a blastocyst.

  In order to confirm whether or not the DNA encoding EGFP has been introduced into the above-obtained individual, RNA is extracted from the body tissues of offspring 1 and 2 that are offspring, and EGFP or KO is specifically detected. The following primer sets were used for genotyping by RT-PCR. As endogenous control, β-actin was compared by RT-PCR using cDNA generated from the skin of wild type marmoset as negative control.

10 μL of the PCR mixture was prepared using 1 × PCR buffer (10 mM Tris-HCl [pH 9.0], 50 mM KCl), 2.5 mM MgCl ₂ , 0.2 mM dNTPs, 10 μM each primer set, and It was made by mixing 0 U of KOD-PLUS-NEO polymerase. The amplification process was performed under the following conditions: heat treatment at 94 ° C. for 2 minutes; annealing at 98 ° C. for 10 seconds, annealing at 63 ° C. for 30 seconds, elongation at 68 ° C. for 30 seconds as one cycle; Each primer set was as follows. The results of RT-PCR are shown in FIG.

EGFP:
5'-CAAGGACGACGCGACACTACAAGACC-3 '(SEQ ID NO: 19) (forward primer)
5'-GCTCGTCCATGCCCGAGAGTGA-3 '(sequence number 20) (reverse primer)
KO:
5'-GAGGGCCTGAGTCGGAGA-3 '(sequence number 21) (forward primer)
5'-CACCAGCCTGTGGCCGATGA-3 '(sequence number 22) (reverse primer)
β-actin:
5'-TGGACTTCGAGCAGGAGAT-3 '(sequence number 23) (forward primer)
5'-CCTGCTTGCTGATCCACATG-3 '(sequence number 24) (reverse primer)

(result)
In FIG. 5, from the left, wild type (negative control), placentas of pups 1 and 2 respectively, hair roots of pups 1 and 2 respectively, skins of p1 and p2 each, blood of pups 1 and 2 respectively The result of RT-PCR is shown. It was confirmed that EGFP was expressed and KO was not expressed in the placenta of each of Pups 1 and 2 and in the hair root and P1 and P2 of each of P2. Although there was no expression in the hair roots of the skin of the pup 1 and the pup 2, the founder individual itself is usually a mosaic, and the expression level of the promoter may be slightly different depending on the tissue.

[Example 7]
[Target gene introduction experiment-1]
As a target gene to be inserted into mammalian genomic DNA, a mutant (LRRK2 (G2019S)) (hereinafter also referred to as “mutated LRRK2”) in which the glycine residue at position 2019 of LRRK2 causing Parkinson's disease is a serine residue Selected. A region in which the gene DNA encoding EGFP, which is a fluorescent expression reporter, and the gene DNA encoding the above-mentioned mutant LRRK2 upstream thereof are joined at 2A, and further a CAG promoter functioning in non-human primate cells is linked upstream thereof. As the insertion region by the PB transposon. Including the configuration of CMV ₁ promoter-hKO1 d2 PEST-polyA-PB5 'TR-CAG promoter-LRRK2 (G2019S) -2A-EGFP-polyA-PB3'TR added with a foreign gene transfer determination region upstream of PB5'TR A mutated LRRK2-containing PiggyBac circular vector (FIG. 6) was generated.

  In a procedure similar to Example 1, human embryonic kidney 293T cells were cotransfected with the piggyBac transposase circular vector, using the above-mentioned mutant LRRK2-containing PiggyBac circular vector instead of the KO / EGFP-PB transposon-containing circular vector. With respect to the transfected 293T cells, EGFP was observed over time using an excitation light of 488 nm, and KO1 and EGFP were observed over time with an inverted fluorescent microscope (manufactured by Olympus Corporation) using excitation light of 548 to 584 nm. The results are shown in (a) and (b) of FIG.

(result)
As is clear from FIG. 7 (a), in the negative control cells (a cells) to which only the mutated LRRK2-containing PiggyBac vector was added (Day 0), expression of EGFP and KO1 started on Day 1. Also on Day 5, it can be confirmed that both EGFP and KO1 are strongly expressed. However, on Day 10, both of the fluorescence by EGFP and the fluorescence by KO almost disappeared.

  On the other hand, as is clear from FIG. 7 (b), in the day 0 cells (b cells) cotransfected with the PiggyBac vector containing the mutated LRRK2 and the piggyBac transposase mRNA, expression of EGFP and KO1 was observed on Day 1 Although it can be performed, there are cells (blue arrows) in which the green fluorescence of EGFP is strong and the intensity of the orange fluorescence of KO1 is small compared to the above (a) cells on Day 5; It was confirmed that the presence of (white arrow) and the cells expressing both EGFP and KO1 were mixed. Therefore, when EGFP is introduced into genomic DNA by the action of transposase, the expression of EGFP is stabilized and enhanced while the expression of KO1 not introduced into genomic DNA is rapidly attenuated (by around Day 5) That was confirmed. On Day 10, the fluorescence of KO1 was not observed and many cells expressing only EGFP were observed, confirming that the piggyBac transposon region containing LRRK2 mutant gene DNA and EGFP was introduced into the genomic DNA of 293T cells. The

[Example 8]
[Target gene introduction experiment-2]
(Foreign gene transfer to mouse neurosphere)
The following vector was constructed as a target gene for inserting a large gene into mammalian genomic DNA. First, downstream of G protein-coupled receptor 56 (gpr56) minimal promoter (SEQ ID NO: 25) (Bae et al., Science 2014 etc.) known to selectively have activity in neural stem cells and neural progenitor cells The fluorescent reporter gene EGFP was conjugated to the luminescent reporter Luciferase 2 (second expression cassette). However, since the above gpr56 minimal promoter has no activity in embryo, the first expression cassette of the EOS promoter and reporter gene hKO1 having activity in embryo as a marker that can select the introduced part in embryo is downstream of the second expression cassette After placement, the two cassettes were joined by an insulator to form a PB transposon insertion area (PiggyBac transposon area). In the determination region upstream of PB3 ', Cerulean (SEQ ID NO: 15) expressing blue fluorescence not used in the insertion region is joined with d2PEST and placed under the CMV promoter, and CMV ₁ promoter-Cerulean-d2PEST-poly A PB transposon circular vector shown in FIG. 8 containing A-PB5'TR-hgpr56-eGFP-luciferase-Ins-EOS-hKO1-polyA-PB3'TR was prepared. In the above, Ins means an insulator. In this experiment, the PiggyBac transposon region is used to prevent interference between the cassettes, as it has two cassettes, each containing a promoter and a foreign protein.

  Hgpr56e1m-EGFP was prepared using a PB transposon circular vector in which Cerulean was placed in the above-mentioned foreign gene transfer determination region and the gpr56 minimal promoter was loaded in the transposon region, and a piggyBac transposase vector (Medical Research Council (UK)). Attempts were made to introduce DNA encoding Luciferase 2 (second expression cassette) and EOS-hKO1 (first expression cassette) into genomic DNA of mouse neurosphere.

(Formation of mouse neurosphere)
Neural stem cells and nerves in suspension culture method of mouse embryonic brain striatum in MHM medium (Murayama et al., J Neurosci Res 2002) supplemented with bFGF (basic fibroblast growth factor), EGF (epidermal growth factor) and B27 Progenitor cells were expanded to form neurospheres.

(Co-transfection into mouse neurosphere)
The PB transposon-containing circular vector carrying the gpr56 minimal promoter and the piggyBac transposase circular vector were electroporated into the mouse neurosphere at a concentration ratio of 2: 1 (Day 0). A neurosphere transfected with only the PB transposon-containing circular vector without transposase was used as a negative control. With regard to the transfected neurosphere, Cerulean using excitation light 452 nm, EGFP using excitation light 488 nm, hKO1 using excitation light 548 to 584 nm, temporally with an inverted fluorescence microscope (manufactured by Olympus) I observed it. In addition, Beetle Luciferin, Potassium Salt (manufactured by Promega) was added to the neurosphere of Day 5, and the expression of luciferase was observed by IVIS Spectrum In Vivo Imaging System (manufactured by Perkin Elmer). The results are shown in FIG.

(result)
As apparent from FIG. 9, in the negative control cells to which only the PB transposon-containing cyclic vector was added (FIG. 9 (a)), Cerulean in the judgment region and KO in the insertion region were expressed in the same cell in Day 1 and Day 5 On the other hand, in the section into which transposase was introduced, cells expressing KO were seen in cells in which expression of Cerulean in the judgment area was not observed on Day 5. On the other hand, although expression of EGFP conjugated to the gpr56 minimal promoter was not observed in either section, luminescence of its downstream luciferase was observed only in the section into which transposase was introduced.

  Therefore, when DNA encoding gpr56-EGFP-Luciferase 2 and EOS-hKO1 is introduced into genomic DNA by the action of transposase, while the expression of hKO1 is stabilized and enhanced in cells with EOS promoter activity, It was confirmed that the expression of Cerulean not introduced into the genomic DNA was rapidly attenuated (by around day 5). In addition, the transposon region was introduced into the genomic DNA of neurosphere, as the expression of a luminescent reporter under gpr56 having activity in neural stem cells and neural progenitor cells was observed only when cotransfected with transposase. That was confirmed.

[Example 9]
Using 7 μg / μL of the above gpr56-EGFP-Luciferase 2 and EOS-hKO1-containing piggyBac vector and 3.5 μg / μL of mRNA prepared from the piggyBac transposase expression vector using the micromanipulator (manufactured by Leica) as in Example 1. It was microinjected into the male pronucleus of mouse embryos. Changes in fluorescence expression during embryo development were observed with an inverted fluorescence microscope (manufactured by Olympus), and the results are shown in FIG.

(result)
As apparent from FIG. 10, the orange fluorescence of hKO1 is obtained in the embryo (orange embryo indicated by the white arrow) in which gpr56-EGFP-Luciferase2 and EOS-hKO1 are inserted into the genomic DNA by the piggyBac transposon mechanism acting. After 24 hours (Day 1), the blue fluorescence of Cerulean was weakly observed on Day 1 (blue embryo indicated by red arrow), but then the expression disappeared and after 96 hours while hKO1 alone fluorescence was sustained It was confirmed that development progressed to blastocysts (Day 4) (FIG. 10). Therefore, when the insertion region by PB transposon is introduced into genomic DNA by the action of transposase, the expression of hKO1 is stabilized and enhanced, while the expression of Cerulean upstream of PB3 'not introduced into genomic DNA is rapid. It was confirmed that the Although EGFP and Luc2 are not observed in mouse embryos because the Gpr56 minimal promoter has no activity in embryos, expression of embryo-active EOS-hKO, which is carried in the same PB transposon insertion region, has been confirmed. It was confirmed that the piggyBac transposon region was introduced into mouse embryonic genomic DNA.

Claims (9)

A vector for foreign gene transfer into a mammalian cell, comprising (A) a foreign gene transfer determination region and (B) a piggyBac transposon region;
The (A) foreign gene introduction determination region codes (a1) a promoter DNA for determination that functions in mammalian cells, and (a2) a fluorescent protein for determination operably linked downstream of the promoter DNA for determination. Containing the DNA
The (B) piggyBac transposon region is disposed between (b1) a pair of piggyBac transposon sequences recognized by the piggyBac transposase added on the 5 'end side and the 3' end side, and the transposon sequences ( b2) A foreign substance characterized by comprising: a foreign protein promoter DNA which functions in mammalian cells; and (b3) a DNA encoding a foreign protein including a fluorescently labeled protein operably linked downstream of the foreign protein promoter DNA. Gene transfer vector. The vector according to claim 1, wherein the foreign protein containing a fluorescently labeled protein further comprises one or more target proteins in addition to the fluorescently labeled protein. The vector according to claim 1 or 2, wherein the foreign gene transfer determination region further comprises (a3) a DNA encoding a protein destabilizing peptide. The vector according to any one of claims 1 to 3, wherein the fluorescence emitted from the fluorescent protein of the foreign gene transfer determination region and the fluorescence emitted from the fluorescently labeled protein are in a complementary color relationship. A vector kit for foreign gene transfer, comprising the piggyBac foreign gene transfer vector according to any one of claims 1 to 4 and piggyBac transposase. A method for selecting a foreign transgenic cell comprising the following steps (a) to (d).
(A) preparing a foreign gene transfer vector according to any one of claims 1 to 4;
(B) preparing mammalian cells;
(Iii) injecting the foreign gene transfer vector prepared in (ii) above and piggyBac transposase into cells;
(D) selecting a cell in which the fluorescence of the fluorescent protein for determination by the vector for foreign gene transfer has been rapidly attenuated as a cell into which the foreign gene has been introduced; The method according to claim 6, wherein the foreign transgenic cell is a fertilized egg. A method for producing a non-human mammalian transgenic animal comprising the step of transplanting a foreign gene-introduced fertilized egg selected by the method according to claim 7 into a non-human mammalian animal temporary mother and obtaining an offspring. A non-human mammalian transgenic animal cell having a foreign gene and having fertility and fertility obtained from the non-human mammalian transgenic animal produced by the method according to claim 8.