JPH1080274A - Tetracycline transactivator bearing nuclear transport signal bonded thereto - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a recombinant gene which can strictly control the gene expression in mammalian cells and can produce protein by including the nuclear localization signal(NLS) gene and the tetracycline transactivator(tTA) gene. SOLUTION: This combinant gene comprises (A) the NLS gene originating from the large T protein of SV40 virus, (B) tTA gene and, preferably, (C) the promoter gene such as CM promoter of cytomegarovirus. The NLS gene and tTA gene is integrated in the inframe state and this recombinant gene preferably bears an extraneous gene coding for proteins, for example, virus envelop protein, hormones, cytokine and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gene expression system in animal cells, and more particularly, to a combination of genes capable of strictly controlling the expression of a transgene, and use thereof.

[0002]

2. Description of the Related Art Substances other than components essential for cell growth are often synthesized inductively when necessary. For this reason, when a foreign gene is introduced into animal cells to produce a certain protein in the cells, the introduced gene is constantly expressed and the protein is synthesized. The substance may affect proliferation and may be toxic to cells.

In order to solve these problems, a metallothionein gene (EK. Mayo et al., Cell 29, 99
-108, 1982), heat shock gene (L. Nouer
Et al., "Heat Shock Response" edited by L. Nouer, published by CRC) and some hormone genes (F. Lee et al., Na.
Nature, vol. 294, pp. 228-232, 1981). However, in these eukaryotic cell-derived promoters, even when not induced, the introduced gene is expressed to a small extent, and a small amount of protein is produced. Prokaryotic cell l
It has been reported that the inducible promoter of the ac gene also works in animal cells (M. Hu et al., Cell 48, 555-555).
566, 1987), but also insufficient to tightly control the production of the gene product of interest.

[0004] As a prokaryotic cell-derived gene expression system other than the lac gene, a tetracycline / gene using a gene encoding a tetracycline transactivator (tTA) controlled by tetracycline (hereinafter also referred to as a tTA gene) developed by Bujard et al. Repressor ( tet R) / Tetracycline operator ( te
The tO ) gene expression system has been reported. tTA bound to tet R cannot bind to tet O in the presence of tetracycline, and gene expression does not occur. Meanwhile, tTA in absence of tetracycline can bind to tet O, expression of the gene occurs. As described above, when tTA is used, the gene expression switch is turned on / off depending on the presence or absence of tetracycline.
Off (ON / OFF) can be adjusted.

[0005] However, at present, even when tetracycline is added to suppress the expression of the transgene, about 0.01% of the expression of the transgene when unrepressed is observed.
Expression is not completely suppressed (M. Gossen et al., Proceed
89 volumes of edings of National Academy of Science, USA
5547-5551, 1992). As described above, at present, no system has been reported that can strictly control the expression of a gene introduced into animal cells.

[0006] On the other hand, in order to prepare a packaging cell line for producing a retroviral vector, gag , pol , and e , which are essential for retrovirus replication, are conventionally prepared.
It was necessary to introduce at least one of genes such as nv into cells. In this case, depending on the position where the introduced vector gene is integrated into the chromosome of the cell, expression may not be completely suppressed even when unnecessary (Leakin
ess), the expression level is often low when needed. Therefore, after transfection, a great deal of effort is required to select an excellent packaging cell line. In addition, changing the retroviral infection specificity by changing the retroviral coat protein requires the production of a vector gene that incorporates the target gene for each gene encoding the coat protein of each virus. Was. Therefore, producing individual vectors was a very laborious operation.

[0007]

Accordingly, one of the objects of the present invention is that expression of a transfected gene does not occur at all or hardly observed when not induced, and does not affect the growth of the transfected cells. However, it is an object of the present invention to provide a gene expression system capable of producing a target protein by expressing an introduced gene upon induction.

[0008] Another object of the present invention is to provide a pseudotype that replaces conventional packaging cell lines.
And a means for easily constructing a retrovirus vector production system.

[0009]

As a result of various studies, the present inventors have found that a nuclear localization signal, NLS (Nuclear Localiza), is used in the gene expression system using tetracycline of Bujard et al.
It has been found that the above-mentioned object can be achieved by incorporating a gene for the signal. In other words, roughly speaking,
Viruses comprising a combination of the NLS gene and a gene encoding the tetracycline transactivator (tTA) (tTA gene), and further comprising a gene with a promoter gene incorporated therein, have a gene enhanced in the absence of tetracycline. Expression was found, but in the presence of tetracycline, gene expression was found to be substantially 100% suppressed.

Therefore, according to the present invention, there is provided a gene comprising the NLS gene and the tTA gene, as a means for achieving the above object. In a preferred embodiment for achieving the object of the present invention, the above-mentioned NLS gene and tTA gene are in-frame, that is, a gene into which a protein which is a gene product thereof is integrated so as to exert a desired action. Can be As long as these gene products have the desired effect,
The position where each is incorporated is not limited.

[0011] Also provided are genes into which a promoter gene, which is essential for expressing these genes, is further incorporated. Usually, the promoter is the NLS
The gene and the tTA gene are preferably integrated so as to be located upstream of the gene. According to the present invention, any promoter capable of expressing the above-mentioned NLS gene and tTA gene can be used regardless of its origin and type. However, without limitation, NLS genes that can be conveniently used for the purposes of the present invention include the large T protein (or antigen) of the SV virus
And promoters include a cytomegalovirus CM promoter, a cytomegalovirus enhancer and a mouse Moloney leukemia virus promoter-coupled Rx promoter,
Examples include the CA promoter in which a cytomegalovirus enhancer and a β-actin promoter are linked, and the Tet promoter of a tetracycline gene.

According to the present invention, the above-mentioned recombinant gene comprises:
A gene encoding a foreign protein may be further incorporated. As described in detail below, the recombinant gene of the present invention can strictly control its expression depending on the presence or absence of tetracycline, for example, when growing cells containing such a gene, the expression of any of the above-described integrated gene Has an adverse effect, not only can its expression be substantially suppressed 100%, but also its expression can be enhanced in some cases.

Accordingly, an expression system suitable for producing a specific protein for any purpose is provided. Therefore, the foreign protein referred to in the present invention may be any protein according to each purpose. Examples of such foreign proteins include, but are not limited to, viral coat proteins, hormones, cytokines, and receptors for hormones and cytokines.

The above-described recombinant gene is conveniently carried on a certain vector and used in the form of a plasmid. Specific examples of the plasmid according to the present invention will be given in the Examples below, which are representative examples.Plasmids that can be easily assumed by those skilled in the art from these representative examples are all within the scope of the present invention. . According to the present invention, there is also provided a virus containing the above-mentioned recombinant gene. For the purposes of the present invention, preferred viruses are those that use adenovirus as the host virus. Adenoviruses can be conveniently used with both the mammalian mastadenovirus and the bird avian adenovirus, but more preferably the former.

It is preferable that the NLS gene, the tTA gene and the foreign gene are incorporated downstream of the promoter.

According to the present invention, there is also provided an animal cell transfected (infected) with the above virus. Preferred animal cells include mammalian cells infected by the mast adenovirus according to the present invention, specifically A375 (ATCC CRL) which is a human melanoma cell line.
-1619), a human brain tumor cell line T98 (ATC
C CRL-1690), A172 (ATCC CRL)
-1620), U373 (ATCC HTB-17) and mouse 3T3 cells (ATCC CCL-163). By growing the cells thus obtained, for example, foreign protein can be efficiently produced.

Each of the above-mentioned subjects of the present invention can be specifically obtained according to the following embodiments or the following series of operations.

For example, first, a tetracycline transactivator (tTA) is introduced into a cytomegalovirus promoter (CM promoter, M. Gossen et al., Pro.
ceedings of National Academy of Science, USA 89
Vol., Pp. 5547-5551, 1992). Also,
tTA gene expression reporter for detecting activation ability (Reporter) lac Z gene allows control by tetracycline tetracycline promoter is a gene (Tet promoter, M.Gossen et al, Proceedings
of National Academy of Science, USA 89, 55
(See pages 47-5551, 1992). Animal cells are co-infected with these two types of adenovirus simultaneously (Cotransfecti
on) to perform gene transfer. At that time, two types of recombinant adenoviruses of tTA alone (the same system as Bujard et al.) And NtTA with a nuclear localization signal (NLS) added to tTA were prepared, and both of them had the ability to suppress gene expression in the presence of tetracycline. To compare and examine the ability to induce gene expression in the absence of tetracycline. Lac Z As a result, in the tetracycline system of tTA in absence
While the gene expression is about 30%, NtTA
In the system (1), the gene expression was enhanced by 80% or more and 2.7 times or more.

On the other hand, NtTA in the presence of tetracycline
In the system using, gene expression is suppressed by 100%, whereas in the system using tTA, gene expression is suppressed by only about 50%. Through the above operations, those skilled in the art will be able to reach the present invention by confirming the action of each recombinant gene.

According to the present invention, there is further provided use of the above virus in combination with another virus. For example,
An adenovirus containing a suitable promoter and a gene incorporating NtTA (referred to as the first virus) is
A recombinant adenovirus containing a gag - pol gene involved in retrovirus replication, which is incorporated downstream of a tetracycline promoter (Tet promoter) controllable by tetracycline (secondary gene)
And a recombinant adenovirus comprising a gene encoding a retroviral coat protein downstream of the Tet promoter (referred to as a third virus). I will provide a.

For example, a retrovirus high expression system can be provided by infecting animal cells with the above virus group. In addition, specific animal cells are co-infected with the first, second and third viruses, while co-infected with the first and second viruses, the gene transfer efficiency in each cell is detected, and When the transfection efficiency is similar to that of the former cells (including a case where the efficiency is slightly reduced), it can be inferred that the cells used for infection have been infected by any retrovirus.
Therefore, according to the present invention, it is also possible to detect whether or not an animal cell has been infected by a retrovirus.

[0022]

Now, the present invention will be described in further detail with reference to specific examples.

Example 1 Plasmid pUHD15-1 (M. Gossen et al., Proceedin
gs of National Academy of Science, USA 89, 5
547-5551, 1992), XhoI and H
Cleavage with indIII produced a 2.3 kb CMtTA fragment. After blunting the end of this fragment, SwaI (restriction enzyme Swa) of plasmid pAxcw was used.
I was purchased from Boehringer Mannheim and all other restriction enzymes were New England unless otherwise noted.
Cosmid pAxCMtTA was prepared by inserting it into a site (purchased from Bio-Lab), and the method of Saito et al.
yake et al., Proceedings of National Academy of Scienc
e, USA, 93, 1320-1324, 1996).
A recombinant adenovirus (Ax) by transfecting human 293 cells (ATCC CRL-1573) with TPC (terminal peptide complex);
CMtTA) (see FIG. 1).

The tTA gene converts pUHD15-1 to Xba
After cutting with I, the fragment was converted to Blunt end and then cut with BamHI to cut out a fragment of about 1 kb. Also, the pRx.nZpA gene is a plasmid pRx
XR30-PK sequence (--- CC ATG GAT AAA GCT GAA) synthesized at the 5 'end of LacZ sequence of LacZ (obtained from Dr. Wakimoto [Cancer Research Association Cancer Chemotherapy Center])
TTT CTC GAA GCT CCTAAG AAG AAA CGT AAG GTA GAA GA
T CCT AGG AAT TC ---, D. Kalderon et al., Cell 39,
Page 499-509, 1984) to produce plasmid pRx.nZ, followed by plasmid pRx.n.
Z was cut with BamHI and HindIII, and the 3′-terminal LTR fragment was replaced with the poly (A) sequence of SV40, which is a fragment obtained by cutting about 470 bp of BamHI and HindIII derived from plasmid pUHD15-1. did. pRxNtTA is the plasmid pRx · nZ
After cutting pA with EcoRI, it is Blunt-end and further B
It was prepared by connecting the above-mentioned tTA gene to the site cut with amHI. In this plasmid, XR30-
The nuclear translocation signal (NLS) of PK is linked to the tTA gene so that it is in frame with the N-terminus of the protein encoded by the tTA gene.

The plasmid pTetZ is identical to the plasmid pRx
-About 3.1 kb NLS-la obtained by cutting nZ (obtained from Dr. Wakimoto (above)) with XbaI and BamHI.
The cZ fragment was converted to plasmid pUHD10-3 (M. Gossen
Et al., Proceedings of National Academy of Science, US
A, Vol. 89, pp. 5547-5551 (1992)) at the XbaI and BamHI sites. This p
After cutting TetZ with XhoI and HindIII, T4
DNA polymerase (New England Bio
(Purchased from Labo) to Blunt end. This about 4.
The 1 kb fragment was converted into pAx by the method of Saito et al.
A recombinant adenovirus (AxTetZ) having the lacZ gene inserted into the SwaI site of cw was prepared (see FIG. 1).

The plasmid pTetNtTA was prepared by inserting an approximately 1.1 kb fragment obtained by cutting the plasmid pRxNtTA with NcoI and BamHI into the NcoI and BamHI sites of the plasmid pTetZ. Plasmid pCMNtTA was constructed from plasmid pUHD15-1 to E.
After cutting with coRI, Blunt end and Xho
The 0.77 kb CM promoter fragment obtained by digestion with I.I.KpnI of the previously prepared plasmid pTetNtTA was used.
It was prepared by cloning into a site cut with XhoI after Blunt end after cleavage. CMNtTA
Recombinant adenovirus expressing the gene (AxCMN)
tTA) is obtained by combining the CMNtTA gene with XhoI and Hind.
III, cut out as a 2.3 kb fragment, blunt-ended with T4 DNA polymerase, cloned into the SwaI site of cosmid pAxcw to prepare cosmid pAxCMNtTA, and then prepared by the method of Saito et al. reference).

The prepared recombinant adenovirus AxCM
tTA and recombinant adenovirus AxCMNtTA
When HeLa cells were infected in the range of OI = 10 to 1250, the system using an adenovirus carrying NtTA in the absence of tetracycline at an MOI of 250 was better than the system using an adenovirus carrying tTA in the absence of tetracycline. About 2.7-fold gene expression was observed. In the presence of tetracycline, gene expression was completely suppressed in the system using the adenovirus carrying NtTA, whereas only about 50% was observed in the system using the adenovirus carrying tTA. None (see FIG. 2).

Example 2 Preparation of plasmid pCANtTA
CAGGS (H. Niwa et al., Gene 108, 193-20)
0, 1991), cut with SalI and HindIII, and then converted to Blunt end. Then, a plasmid pCAcc was prepared by coupling a ClaI linker. Then, the plasmid pCacc was cut with EcoRI, and the plasmid pTetNtTA was added to the Blunt end site. KpnI and H
After cutting with indIII, a fragment of about 1.5 kb, which was made into a Blunt end, was inserted and inserted. An approximately 2.7 kb fragment obtained by cutting the plasmid pCANtTA with ClaI was inserted into the ClaI site of the plasmid pAxcw by the method of Saito et al. To prepare a recombinant adenovirus AxCANtTA (see FIG. 1).

The plasmid pUHD15-1 was digested with XbaI and then blunt-ended, and then an approximately 1 kb tTA fragment digested with BamHI was prepared. This tTA fragment was constructed using the plasmid pRx.nZpA prepared in Example 1 as E
After digestion with coRI, the fragment was converted to Blunt end, and further inserted into the site digested with BamHI to prepare a plasmid pRxNtTA. Plasmid pRxNtTA was replaced with XhoI and Hin.
The about 3.6 kb fragment cut with dIII and made into Bluntend was ligated to the plasmid pAxc by the method of Saito et al.
recombinant adenovirus A inserted into the SwaI site of w
xRxNtTA was produced (see FIG. 1).

The plasmid pRxNtTA was replaced with NcoI and B
The approximately 1.1 kb fragment cut with amHI was inserted into the NcoI and BamHI sites of the plasmid pTetZ prepared in Example 1 to prepare pTetNtTA, and the SwaI of pAxcw was prepared by the method of Saito et al.
Insertion into the recombinant adenovirus Ax
TetNtTA was produced (see FIG. 1). The prepared recombinant adenoviruses AxCANtTA, AxCMNtT
A, AxRxNtTA and AxTetNtTA are assigned MOI =
When HeLa cells were infected at 250, the system using the CA promoter in the absence of tetracycline was 8
5.5% gene expression was observed, 82.7% gene expression was observed using the CM promoter, 82.4% gene expression was observed in the system using the Rx promoter, and the system using the Tet promoter was observed. Showed 39.9% of gene expression. In the presence of tetracycline, gene expression was completely suppressed in all four. When these results and the result of Example 1 are summarized, as shown in Table 1, CA,
The CM and Rx promoters showed almost the same gene expression activity, while the Tet promoter showed only about half the gene expression activity. However, NtTA
The gene expression activity was higher in the system using CMtTA than in the system using CMtTA (see FIG. 3).

TABLE 1 Comparison of transgene expression efficiency with nuclear localization signal (NLS) and various promoters. Gene expression system AxCANtTA AxCMNtTA AxRxNtTA AxTetNtTA AxCMtTA expression efficiency (%) 85.5 82.7 82.4 39.9 29.2 Gene expression in the presence of tetracycline in each cell transfected with each adenovirus prepared was NtTA
Is completely suppressed at a concentration of 1 μg / ml or more in tetracycline, whereas it is not completely suppressed at a concentration of 100 μg / ml of tetracycline in a system of tTA. Further, in the absence of tetracycline, CMNtTA, CANtTA and RxNtTA systems show a transgene expression efficiency of 80% or more,
Only about 25% expression efficiency was observed in the CMtTA system (see FIG. 4).

[0032]Example 3 Recombinant adenovirus AxCMt prepared in Example 1
TA or AxCMNtTA and AxTetZ to HeLa
After co-infection of cells, in the presence of tetracycline or
Β-Gal staining was performed after culturing in the absence of
Of course, in the system of tTA (AxCMtTA and AxTetZ)
In the absence of tetracycline, approximately 30% of the total cells
Although only the cells were stained, NtTA (AxCMNtT
A and AxTeTz) systems account for almost 100% of the total cell number.
Cells stained. Also, in the presence of tetracycline
In the NtTA system, β-Gal was completely generated without staining.
While the current is suppressed, some in the tTA system
(15%) cells were stained and β-Gal expression was 50%
To a lesser extent (see FIGS. 5 and 6). Example 4 Plasmid pSKII + VSVG is transformed into plasmid pLG
RNL (N. Emi et al., Journal of Virology 65, 12
02-1207, 1991) cut with BamHI
The fragment was prepared using a fragment of about 1.6 kb. Plasmi
PTetVSVG is the plasmid pSKII + VSV
About 1.6 kb fragment obtained by cutting G with BamHI was added.
Made by inserting into the BamHI site of midpUHD10-3
did. Plasmid pTetVSVG was transformed with XhoI and Hin.
After cutting with dIII, about 2.4 kb of Blunt end
The fragment was purified by the method of Saito et al.
Recombinant adenovirus AxTetV inserted into I site
SVG was produced (see FIG. 1).

The prepared recombinant adenovirus AxTe
HeLa cells were co-infected with both tVSVG and AxRxNtTA, and immunostaining was performed using a monoclonal antibody against VSVG (clone P5D4, Sigma # V5507). Almost 100% of the cells showed expression of the VSVG gene product. . In contrast, when HeLa cells were infected with any one of the above recombinant adenoviruses, expression of the VSVG gene product was not observed in any case.

[0034] Example 5 purpose of monitoring the production efficiency of retroviral vectors, retroviral vectors MFGlacZ that express the reporter gene lac Z (Dranoff et al., Pr
oceedings of National Academy of Science, USA 90
Vol., Pp. 3539-3543, 1993).
5, U373, T98G, A172, etc. to infect human cultured cells, and A375 / Z, U373 / Z, T
Transgenic cell lines such as 98G / Z and A172 / Z were obtained. lac Z gene transfer efficiency, expression detection of lac Z by flow cytometer, as well as detection by X-gal staining and evaluated for parallel. A375, U373, A
172, T98G both with high efficiency of 50% to 100%
A transgenic strain of the lac Z gene was obtained.

The plasmid pTetGP is a MoMLV
(Schnich et al., Nature 293, 543-548,
1981)), the 5307 bp gag - pol gene was transformed from nt563 (HindIII) to nt5870.
Up to (ScaI) and cut out with HindIII / ScaI, and the Tet promoter cut with XhoI to Blunt end, and cut out with EcoRI, were cloned into the SpeI and BglII sites of pCAcc. (However, actually, the gag - pol of MoMLV is divided into fragments, subcloned into pBluescript SKII, and further subcloned into pCAcc to obtain pCA.
GP in multiple steps, replacing the CA promoter with the Tet promoter.
GPs have been produced. ) Cosmid pAxTetGP
Was prepared by cloning from pTetGP into ClaI. Furthermore, the cosmids pAxTetGP and Ad5.
293 cells were co-infected with DNA-TPC to produce recombinant adenovirus AxTetGP.

The prepared recombinant adenovirus AxTe
t375, AxTetGP and AxCANtTA, or A375 /
Z cells, T98 / Z cells, A172 / Z cells and U3
73 / Z cells were co-infected, and two days later, a culture supernatant presumably containing retrovirus was collected. Using a culture supernatant which is thought to contain this retrovirus, 8 μg / m 2 was added to monitor cells (such as NIH 3T3).
Infection was carried out in the presence of 1 Polybrene (Polybrene <Sigma>), and after 2 days, X-gal staining was performed to calculate the retroviral infection efficiency. As a result,
A375 / Z cells, T98 / Z cells and U373 / Z
10 ⁵ to 10 ⁶ pfu / ml of high titer La from cells
It was shown that a Pseudotype retrovirus incorporating the cZ gene could be recovered. On the other hand, the retrovirus-producing ability of A172 / Z cells was low. Using this method, a cell line having a high retrovirus-producing ability can be easily selected. Also, A375 / Z cells produced retrovirus without simultaneous infection with the adenovirus AxTetVSVG. This indicates that A375 cells already have the gene for the env- like protein required for retrovirus replication in the cells.

Table 2: VSVG pseudotype retrovirus gene transfer efficiency Gene transfer efficiency (%) A375 / Z T98 / Z A172 / Z U373 / Z MOCK 0 0 0 0 TetVSVG alone 0 0 0 0 TetGP alone 0 0 0 0 CANtTA alone 0 0 0 0 TetVSVG + TetGP 0 0 2/0 * 0 TetVSVG + CANtTA 3 0 0 0 TetGP + CANtTA 42.0 ± 6.66 0.83 ± 0.983 1.5 ± 0.84 2.0 ± 0.89 CANtTA + TetVSVG + TetGP 71.3 ± 5.92 38.7 ± 5.85 5.3 ± 2.80 46.8 ± 6.94 * Only one of the two plates Gene expression was observed

[Brief description of the drawings]

FIG. 1 schematically shows a recombinant gene according to the invention, based on gene translation units, in comparison with known genes.
The abbreviations in the figures have the following meanings. CM: cytomegalovirus promoter CA: promoter combining cytomegalovirus enhancer and β-actin promoter Rx: promoter combining cytomegalovirus enhancer and mouse Moloney leukemia virus promoter Tet: tetracycline promoter tTA : Tetracycline transactivator NLS: Nuclear localization signal pA: PolyA signal

FIG. 2 shows a recombinant gene expression system of the present invention and a known Buj
1 is a graph showing the expression efficiency of a gene expression system by ard et al. In the graph, circles indicate CMNtTA (the present invention).
And the squares are results according to CMtTA (comparison). The open symbols indicate the expression efficiency in the absence of tetracycline, and the filled symbols indicate the expression efficiency in the presence of tetracycline.

FIG. 3 is a graph showing changes in gene expression efficiency depending on the type of promoter in the gene expression system of the present invention. In the graph, circles indicate the expression efficiency of the NtTA transgene by the CA promoter, squares indicate the Rx promoter, and triangles indicate the expression efficiency of the NtTA transgene by the Tet promoter. In addition, a white mark indicates the absence of tetracycline, and a black mark indicates the presence of tetracycline.

FIG. 4 shows a gene expression system according to the present invention (CMNtTA: inverted triangle; CANtTA: horizontal triangle; TetNtT)
A: triangle mark; RxNtTA: square mark) and Comparative Example (tetZ)
FIG. 10 is a graph showing the change in the gene expression efficiency with the change in tetracycline concentration with respect to (alone: diamond; CMtTA: circle).

FIG. 5 is an electron micrograph instead of a diagram showing the morphology of β-Gal stained cells (organisms) showing the expression status of the lacZ gene in the gene expression system of the comparative example (each infected cell). It is. A and b correspond to TetZ alone (comparison), c and d correspond to CMtTA (comparison), respectively, the former (a, c) in the absence of tetracycline, while the latter (b, d) in the presence of tetracycline. Of cultured cells.

FIG. 6 shows β-Gal indicating the expression status of the lacZ gene in the gene expression system (each infected cell) according to the present invention
It is an electron micrograph instead of the figure which shows the form of the stained cell (organism). E and f are respectively CMNtT
A, g and h are CANtTA, i and j are RxNt
TA, and k and l correspond to TetNtTA,
Each of the former (e, g, i, k) is a cultured cell in the absence of tetracycline, while the latter (f, h, j, l) is a cultured cell in the presence of tetracycline.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location // (C12N 5/10 C12R 1:91) (C12N 7/00 C12R 1:92)

Claims (18)

[Claims] 1. Nuclear localization signal (NLS) gene and tetracycline transactivator (tTA)
A recombinant gene comprising the gene. 2. The recombinant gene according to claim 1, wherein the NLS gene and the tTA gene are integrated in frame. 3. The recombinant gene according to claim 1, wherein the NLS gene is derived from SV40 virus large T protein. 4. The recombinant gene according to claim 1, further comprising a promoter gene. 5. The promoter gene is a CM promoter of a cytomegalovirus, an Rx promoter in which a cytomegalovirus enhancer is linked to a mouse Moloney leukemia virus promoter, and a CA promoter in which a cytomegalovirus enhancer is linked to a beta-actin promoter. 5. The recombinant gene according to claim 4, wherein the recombinant gene is selected from the group consisting of Tet promoter of tetracycline gene. 6. The recombinant gene according to claim 5, further comprising a gene encoding a foreign protein. 7. The recombinant gene according to claim 6, wherein the foreign protein is at least one selected from the group consisting of viral coat proteins, hormones, cytokines, and hormone and cytokine receptors. 8. A plasmid carrying the recombinant gene according to claim 1. 9. A virus comprising the plasmid according to claim 8. 10. The virus according to claim 8, wherein the host virus is an adenovirus. 11. N incorporated in an in-frame state
11. A recombinant gene comprising a LS gene and a tTA gene integrated downstream of the promoter.
The virus described. An animal cell transfected with the virus according to any one of claims 9 to 11. 13. The animal cell according to claim 12, wherein the cell to be transfected is a mammalian tumor cell. 14. The virus according to claim 11, wherein the gad - pol gene involved in retrovirus replication is a recombinant adeno containing a recombinant gene incorporated downstream of a tetracycline promoter (Tet promoter) controllable by tetracycline. A group of viruses combined with a recombinant adenovirus comprising a recombinant gene in which the genes encoding the viral and retroviral coat proteins are integrated downstream of the Tet promoter. 15. The virus group according to claim 14, wherein the coat protein of the retrovirus is a transmembrane protein of vesicular stomatitis virus. 16. An animal cell co-infected with the virus group according to claim 14 or 15. 17. A method for producing a retrovirus, comprising co-infection of animal cells with the group of viruses according to claim 14 or 15, and propagating the animal cells thus obtained. 18. A method for detecting a tendency to produce a retrovirus by co-infection with an animal cell suspected of being infected with a retrovirus by one or more of the virus groups according to claim 14 or 15. A method for detecting a retroviral infection state of a characteristic animal cell.