JPH06209780A - Phenotypic tranformation of animal cell and vector therefor - Google Patents

Abstract

PURPOSE:To provide a new method introducing a manifested vector into animal cells and capable of manifestation cloning easily and accurately, and to obtain the vector required for the method. CONSTITUTION:The subject method for phenotypic transformation of animal cells comprises transfecting animal cells with a single chair cyclic DNA or a bacteriophage containing single chain cyclic DNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for introducing a gene into animal cells and a plasmid vector used therein. By this method, for example, a cDNA that can be used to mass-produce a protein useful as a medicine can be easily obtained.

[0002]

2. Description of the Related Art Bioactive proteins produced by cells are
It is widely used in industry such as medicines, diagnostics, biosensors and bioreactors. Advances in genetic engineering technology have facilitated the search for these proteins and enabled their mass production. The basis of this is cDNA cloning technology.

Information on the amino acid sequence of a protein is encoded by mRNA. Therefore, if the mRNA is replaced with DNA, that is, cDNA (complementary DNA) is synthesized, the primary structure of the protein can be determined based on this, and the protein can be mass-produced. Therefore, many so-called cDNA cloning techniques have been developed in which mRNA is extracted from cells, cDNA is synthesized using this as a template, and the cDNA encoding the target protein is extracted from the obtained cDNA library.

When the target protein can be purified in a large amount, an oligonucleotide probe is synthesized based on the amino acid sequence of the purified protein, and the target protein is coded by screening a cDNA library. The method of cloning the cDNA is used. However, when it is difficult to purify due to the small amount of protein, a method called "expression cloning" is adopted (see, for example, Experimental Medicine Supplement, "Gene Engineering Handbook", Yodosha, 1991). This method starts with a vector that incorporates a promoter that is expressed in animal cells, such as
pCDM8 [B.Seed, Nature 329: 840-842 (1987)] and pcD2 [HO
kayama and P. Berg, Mol. Cell. Biol. 3: 280-289 (1983)]
Is used to prepare a cDNA library, the obtained cDNA is transformed into an animal cell, and a cDNA expressing a desired activity is cloned. In each case, a large number
After purifying high-purity vector DNA from the cDNA clone,
This is introduced into animal cells by the calcium phosphate method, the DEAE-dextran method, the liposome method, the electroporation method, etc. (see, for example, Experimental Medicine Separate Volume “Gene Engineering Handbook”, Yodosha, 1991), and the desired activity It is a method of searching for a clone expressing. When using this method, a complicated operation of purifying high-purity vector DNA from a large number of cDNA clones was required.

[0005]

The object of the present invention is to provide a new method for introducing an expression vector into an animal cell and a vector used therefor, which enables easy and reliable expression cloning. is there.

[0006]

[Means for Solving the Problems] As a result of intensive studies, the present inventors have found that single-stranded circular DN is easier to prepare than double-stranded DNA.
The inventors have found that animal cells can be transformed with a phage containing A or single-stranded circular DNA, and completed the present invention. That is, the present invention provides a method for transforming an animal cell, which comprises transfecting an animal cell with a single-stranded circular DNA or a phage containing the single-stranded circular DNA. The present invention also provides a plasmid vector having a replication origin of single-stranded phage, a replication origin for E. coli, a replication origin and promoter for animal cells, a cloning site existing downstream of the promoter, and a marker gene that works in animal cells.

The present invention will be described in more detail below.

The present invention is based on the discovery that when single-stranded circular DNA or a phage containing single-stranded circular DNA is introduced into an animal cell, replication and expression occur in the cell. Therefore, instead of double-stranded DNA that is difficult to isolate and purify, single-stranded circular DNA or single-stranded circular DNA that is easy to prepare is used.
A transfection method using a phage containing DNA has become possible.

The phage containing the single-stranded circular DNA can be prepared from the supernatant of the culture medium after infecting Escherichia coli having the plasmid DNA having the origin of the single-stranded phage with the helper phage. Single-stranded circular DNA is a single-stranded circular DN
It can be prepared from A-containing phage by deproteinization such as phenol extraction. Instead of phenol extraction, the phage coat protein denatured by heat treatment or chemical treatment may be used as it is.

To introduce single-stranded circular DNA into animal cells, conventional methods such as calcium phosphate method, DEAE-dextran method, liposome method and electroporation method can be used as they are. With the conventional protocol, the double-stranded DNA portion may be replaced with the single-stranded circular DNA. It is also possible to transfect animal cells by removing phages containing single-stranded circular DNA or E. coli culture supernatant containing them through a filter to remove E. coli, and then directly adding these to the culture medium of animal culture cells. it can.

Any animal cell may be used as long as it functions as a replication origin or promoter incorporated in the vector. When the replication origin or promoter of SV40 is used as in the example, it expresses T antigen.
COS cells are suitable.

The present invention also provides a vector which expresses selectable properties such as drug resistance in animal cells, and which can prepare single-stranded circular DNA or a phage containing single-stranded circular DNA. By using this, after transfecting a vector into an animal cell, it is possible to select only the cell into which the vector has been introduced and has become drug resistant. The drug resistance gene may be any drug as long as it is expressed in animal cells and exhibits drug resistance, and examples thereof include the neomycin resistance gene and the blasticidin S 2 resistance gene mentioned in the examples. Also, other marker genes such as auxotrophy and temperature sensitivity can be adopted. Examples of the origin of animal cell replication include the origin of replication of SV40. Promoters for expressing the target gene include SV40 early promoter and late promoter, adenovirus late promoter, retrovirus LTR, thymidine kinase promoter, metallothionein promoter, β-actin promoter, and elongation factor promoter. It can be illustrated. The promoter is preferably one that is induced by an external stimulus (in the case of a metallothionein promoter, a heavy metal ion), such as a metallothionein promoter. In addition, a cloning site for inserting the target gene is provided downstream of the promoter. Examples of the origin of single-stranded phage include the origin of f1 phage and the origin of M13 phage. Examples of the replication origin for Escherichia coli include the origins of pBR322 and pUC type plasmids. In addition to this, it may have a drug resistance marker for E. coli, but when the drug resistance gene for animal cells also functions as a drug resistance marker for E. coli as in the case of the example, a drug resistance marker for E. coli is used. Need not be provided separately. Therefore, in the case of the vector of the Example, the blasticidin S resistance gene also functions as a drug resistance marker for Escherichia coli, so the ampicillin resistance gene may be removed.

The structure of pKA1M mentioned in the examples is shown in FIG. Since it has the same cloning site as pKA1 (described in Japanese Unexamined Patent Publication No. 4-117292), the mR
A cDNA library can be prepared from NA.

The minimum condition for the single-stranded circular DNA vector used for transfection is that it has a promoter that works in animal cells before the gene to be expressed. Therefore, even if it is a vector for expressing an animal cell that has been conventionally used, if it has the origin of a single-stranded phage, single-stranded circular DNA
Alternatively, since a phage containing single-stranded circular DNA can be prepared, it can be introduced into animal cells by the method of the present invention. For example, pCDM8 and pKA1 correspond to this (although they do not have a drug resistance gene expressed in animal cells, selection by drug resistance cannot be done).
Therefore, the transformation method of the present invention is not limited to the method using the above-described plasmid vector of the present invention.

[0015]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

The basic manipulations and enzymatic reactions for recombination of DNA are described in the literature ("Molecular Clon").
ing. A Laboratory Manual
l ", Cold Spring Harbor Lab
Oratory, 1989). Unless otherwise specified, the restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo. The buffer composition of each enzyme reaction and the reaction conditions were in accordance with the attached instructions.

Construction of pKA1M (1) Construction of pKA1U Plasmid pUC19 (Pharmacia) 10 μg was added to 100
After digesting with units of ScaI and then 100 units of PstI,
The origin-containing large fragment was isolated and purified from the gel by 0.8% agarose gel electrophoresis. Plasmid pKA1
(Described in JP-A-4-117292) 10 μg is 100 units of Sca
After digestion with I and then 100 units of PstI, a small fragment containing the f1 origin was isolated and purified from the gel by 0.8% agarose gel electrophoresis. After ligation of both fragments, Escherichia coli HB101 was transformed. 10 μg of the plasmid isolated from the transformant was added to 100 units of Pv.
After digestion with uII and then with 100 units of PstI, it was subjected to 0.8% agarose gel electrophoresis to isolate and purify a large fragment containing the origin from the gel.

Plasmid pKA1 (described in JP-A-4-117292)
10 μg was digested with 100 units of AccI, blunt-ended by treatment with Klenow fragment, and then 100 units of PstI.
After digestion with 0.8% agarose gel electrophoresis, a small fragment containing SV40 origin was isolated and purified from the gel. After ligation of both fragments, Escherichia coli HB101 was transformed. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1U. That is, pKA1U is obtained by replacing the pBBR322-derived origin, which pKA1 had, with the pUC-derived origin.

(2) Construction of pKA1UM 10 μg of plasmid pKA1U was digested with 100 units of HindIII, blunt-ended by treatment with T4 DNA polymerase, then digested with 100 units of XhoI, and subjected to 0.8% agarose gel electrophoresis. A large fragment containing the ampicillin resistance gene was isolated and purified from the gel. Plasmid
10 units of pBPV (Pharmacia) with 100 units of EcoRI
Digested with T4 DNA polymerase, blunt-ended and then digested with 100 units of XhoI
A small fragment containing the metallothionein promoter was isolated and purified from the gel by subjecting it to% agarose gel electrophoresis.
After ligation of both fragments, Escherichia coli HB101 was transformed. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1UM.

(3) Construction of pKA1UMbsr 10 μg of plasmid pKA1U was digested with 100 units of Tth111I and then with 100 units of BamHI, blunt-ended by treatment with T4 DNA polymerase, and Escherichia coli HB101 was transformed after self-ligation. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1U-P.

10 μg of plasmid pKA1U-P was digested with 100 units of PstI and then 100 units of BamHI and
The large fragment containing the origin was isolated and purified from the gel by 8% agarose gel electrophoresis. Plasmid pKA1UM1
0 μg for 100 units of PstI, then 100 units of BamHI
After digestion with 0.8%, it was subjected to 0.8% agarose gel electrophoresis, and a small fragment containing the metallothionein promoter was isolated and purified from the gel. After ligation of both fragments, Escherichia coli HB101 was transformed. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1UM-P.

After digesting 10 μg of the plasmid pKA1UM-P with 100 units of BamHI and then 100 units of NotI, 0.8
A large fragment containing the origin was isolated and purified from the gel by subjecting it to% agarose gel electrophoresis. Plasmid pSV2bsr
(Funakoshi Yakuhin) 10 μg was digested with 100 units of PvuII, NotI linker (Takara Shuzo) was added, digested with 100 units of BamHI and 100 units of NotI, and then subjected to 0.8% agarose gel electrophoresis and gel. A small fragment containing the blasticidin S resistance gene was isolated and purified from. After ligation of both fragments, Escherichia coli HB101 was transformed. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1UMbsr.

(3) Construction of pKA1M 1 μg of the plasmid pKA1UMbsr was digested with 10 units of XhoI and then treated with alkaline phosphatase to give 0.8%.
The fragment was isolated and purified from the gel by agarose gel electrophoresis. Plasmid pKA1 (described in JP-A-4-117292) 10
μg was digested with 100 units of XhoI and subjected to 0.8% agarose gel electrophoresis to isolate and purify a small fragment containing the 16S splicing region from the gel. After ligation of both fragments, Escherichia coli HB101 was transformed.
10 μg of the plasmid isolated from the transformant was digested with 100 units of BstXI and then 100 units of KpnI, and
The large fragment was isolated and purified from the gel by 8% agarose gel electrophoresis. After mixing with 10 pmole of each of the two kinds of synthetic oligomers represented by the following formula and annealing,
The ligation reaction was performed and EcoR at the cloning site
V was converted to Eco47III. 5'-GTGGTGAGCGCTCTGGTAC-3 '5'-CAGAGCGCTCACCACCCCC-3' After transformation of Escherichia coli HB101 with the reaction mixture, the plasmid was isolated from the transformant, and the target plasmid pKA1M was determined by restriction enzyme map and nucleotide sequence determination. Was confirmed.

Preparation of single-stranded pKA1M Escherichia coli JM109 was transformed with the plasmid pKA1M, and the transformant was cultured in 3 ml of 2 × YT medium at 37 ° C. for 1 hour and then infected with 100 μl of the helper phage M13KO7 solution. 3 more
After culturing overnight at 7 ° C, centrifuge and take 1.2 ml of the culture supernatant,
To this, 300 μl of 20% polyethylene glycol and 2.5 M NaCl solution was added, and the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged to obtain a phage pellet containing single-stranded DNA. After this was applied to TE, phenol extraction and ethanol precipitation were performed to prepare single-stranded pKA1M.

Transfection of COS cells COS 7 cells (supplied from ATCC) were cultured in 10% FCS-containing DMEM medium at 37 ° C. in a 5% CO ₂ stream until 40 to 50% confluent. The cells detached from the flask by trypsin treatment were suspended in DMEM medium containing 10% FCS, and seeded on a 6-well plastic plate at about 1 × 10 ⁵ cells / well in an amount of 2 ml.
After culturing this for another 22 hours, the medium is removed and DMEM is added.
After washing twice with medium, the DNA sample was added. DNA samples were prepared by dissolving 1 μg of DNA or phage in 0.25 ml of DMEM medium and TRANSFECTAM (IB) in 0.25 ml of DMEM medium.
(Company F) 2.5 μl was mixed and prepared. After adding the DNA sample, incubate at 37 ℃ for 20 hours,
The medium was removed and washed twice with DMEM medium. DMEM containing 10% FCS containing blasticidin S 10 μg / ml.
After adding 2 ml of the medium, the cells were cultured at 37 ° C. in a 5% CO ₂ stream for 1 week. After removing the medium, the number of colonies was counted by Giemsa staining.

The results are shown in Table 1. Double-stranded pK as usual
When A1M was used, about 50 to 100 blasticidin S resistant colonies were obtained. So this plasmid
After being introduced into COS7 cells, it was found that the blasticidin S resistance gene on the plasmid was expressed in the cells. When single-stranded pKA1M or single-stranded pKA1M phage was used as a sample instead of double-stranded pKA1M,
Unexpectedly, resistant colonies were obtained at the same rate as the double-stranded case.

As a control experiment, a similar experiment was carried out on a sample treated with a restriction enzyme. No resistant colonies were obtained from the double strand, but almost the same number of resistant colonies as those from the single strand were not obtained. was gotten. When a sample treated with S1 nuclease, which selectively cleaves single-stranded DNA, was used, the number of resistant colonies obtained from double-stranded DNA did not change, but no colonies were observed from single-stranded DNA. . From the above results, it is clear that the resistant colonies obtained by transfection of single-stranded DNA are not due to the double-stranded DNA contained in the sample in a very small amount, that is, in the case of double-stranded single-stranded DNA. It was shown that it can be transfected into an animal cell as in the above, and that the gene on the single-stranded DNA can be expressed in the cell.

[0028]

[Table 1] Table 1 ─────────────────────── DNA sample colony ─────────────────── ───── Double-stranded pKA1M + restriction enzyme-treated double-stranded pKA1M-S1 nuclease-treated double-stranded pKA1M + single-stranded pKA1M + restriction enzyme-treated single-stranded pKA1M + S1 nuclease-treated single-stranded pKA1M-single-stranded pKA1M phage ＋ ────────────────────────

Plasmid recovery from blasticidin S resistant cells By introducing double-stranded or single-stranded pKA1M,
From cells that have become resistant to blasticidin S, use the Hirt method [Hir
t, B., J. Mol. Biol. 26: 365 (1967)]. For cells in the well, 0.6% SDS, 10 m
0.2 ml of M EDTA was added and left at room temperature for 20 minutes. Transfer the cell lysate to an Eppendorf tube and add 5M NaCl.
50 μl was added and left at 4 ° C. overnight. After centrifugation, the supernatant was taken out, phenol extraction was carried out, and then ethanol precipitation was carried out twice to dissolve the pellet in 10 μl of TE. This liquid 2.5 μ
1 was used to transform E. coli HB101. As a result, a comparable number of transformants were obtained from cells into which either double-stranded or single-stranded pKA1M was introduced. The plasmid isolated from the transformant was the same as pKA1M. From the above, it was shown that the single-stranded pKA1M is replicated in COS7 cells similarly to the double-stranded.

Expression of TNF by pKA1M system Tumor necrosis factor (TNF) cD was inserted at the cloning site of an external gene downstream of the metallothionine promoter of pKA1M.
NA was inserted to prepare a TNF expression vector. Plasmid pKATNF1 (described in JP-A-4-117292) was used in an amount of 100 μg
After partial digestion with 1 unit of NotI and then 10 units of EcoRI, the gel was subjected to 0.8% agarose gel electrophoresis and
A fragment of about 1.7 kbp containing TNF cDNA was isolated and purified. Plasmid pKA1M (10 μg) was added to 100 units of NotI, and then 10
After digestion with 0 units of EcoRI, the fragments were isolated and purified from the gel by 0.8% agarose gel electrophoresis. After ligation of both fragments, Escherichia coli HB101 was transformed. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1M-TNF.

1 μg of single-stranded pKA1M-TNF prepared by infection with helper phage was subjected to CO 2 according to the above protocol.
Transfection of S7 cells was performed. 2 at 37 ° C
After culturing for 0 hour, the medium was removed and the cells were washed twice with DMEM medium. To this, 2 ml of 10% FCS-containing DMEM medium containing no blasticidin S was added and cultured at 37 ° C. for 24 hours in a 5% CO ₂ stream. We did not select with blasticidin S here because blasticidin S already possesses cytocidal activity. Then 0.01
20 μl of M ZnCl ₂ was added and the cells were further cultured for 3 days. When the culture supernatant was taken and the cell killing activity was measured using LM cells, an activity of 10 U / ml was recognized. In addition, when ZnCl ₂ was not added, the activity was 2 U / ml, and it was found that the addition of heavy metal induces the metallothionein promoter. When only pKA1M was introduced, no cytocidal activity was observed regardless of the addition of heavy metals.

[0032]

EFFECTS OF THE INVENTION According to the present invention, expression cloning, which has heretofore required complicated operations, can be carried out easily and reliably. Therefore, by carrying out expression cloning by this method, it is possible to search for cDNA of a protein, which has been difficult in the past.

[Brief description of drawings]

FIG. 1 shows the structure of pKA1M.

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[Procedure amendment]

[Submission date] April 1, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction content]

COS7 cells were transfected with 1 μg of single-chain pKA1M-TNF prepared by infection with helper phage according to the above protocol. After culturing at 37 ° C for 20 hours, the medium was removed and DMEM was added.
Washed twice with medium. To this, 2 ml of 10% FCS-containing DMEM medium containing no blasticidin S was added,
The cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ stream. The selection by blasticidin S was not performed here because blasticidin S already has the cytocidal activity. Then 0.01 M ZnCl ₂ 20μ
1 was added and the cells were further cultured for 3 days. Take the culture supernatant and add L
-Measurement of cell killing activity using M cells yielded 10 U
/ Ml activity was observed. In addition, when ZnCl ₂ was not added, the activity was 2 U / ml, and it was found that the addition of heavy metal induces the metallothionein promoter. When only pKA1M was introduced, no cell killing activity was observed regardless of the addition of heavy metal. Transfection by DEAE dextran method 2 ml of pKA1M / JM109 culture solution as described above
After infection with helper phage, the phage particles obtained by polyethylene glycol precipitation from the culture supernatant were suspended in 100 μl of TE. 2 μl of phage suspension containing 0.4 mg / ml DEAE dextran in DMEM
After mixing with 0.8 ml of medium (adjusted to pH 7.5 with Tris buffer), 10 ⁵ COS-prepared in the same manner as above.
7 cells were added. Then, when blasticidin S was added and cultured in the same manner as above, about 10 blasticidin S-resistant colonies were obtained. Therefore, DE
The AE dextran method was also shown to be capable of transfection with phage particles. The amount of single chain pKA1M contained in the phage suspension used in this experiment was 20 ng. On the other hand, double-stranded pK
When A1M was transfected under the same conditions,
Requires a minimum of 500 ng. Therefore, the efficiency of transfection is higher when phage particles are used. Transfection with E. coli culture supernatant As above, 2 ml of pKA1M / JM109 culture solution
After infection with helper phages in the culture medium, the culture medium was allowed to have a pore size of 0.
After passing through a 22 μm filter to remove the cells, 20 μl of the permeate containing phage was treated with DEA in the same manner as described above.
COS-7 cells were mixed with E-dextran for transfection. As a result, blasticidin S resistant colonies were obtained as in the case of phage particles. That is, it was shown that the E. coli culture solution can be used as it is for transfection. By applying this method, the labor of preparing phage particles can be saved and transfection with a large amount of sample can be easily performed.

Claims (4)

[Claims] 1. A method for transforming an animal cell, which comprises transfecting an animal cell with a single-stranded circular DNA or a phage containing the single-stranded circular DNA. 2. A plasmid vector having a replication origin of single-stranded phage, a replication origin for Escherichia coli, a replication origin and promoter for animal cells, a cloning site existing downstream of the promoter, and a marker gene that works in animal cells. 3. The plasmid vector according to claim 2, wherein the marker gene is a drug resistance gene. 4. The plasmid vector according to claim 3, which is pKA1M.