The present invention relates to a method for stable and high expression of a DNA sequence of interest using a nucleic acid construct that comprises, in a mammalian artificial chromosome vector, the DNA sequence of interest, a matrix attachment region, and a replication origin, and to the nucleic acid construct.
In recent years, the so-called biomedicines, such as proteins or nucleic acids, have been grately expected for use in treatment of diseases that is difficult to cure because of the lack of effective therapy, cancers, and the like. In particular, many antibody medicines have already been used in clinical settings or clinically tested because of their satisfactory effects and few adverse effects, and some of them are ranked high in pharmaceutical sales in the world. Unlike low molecular weight medicines, however, biomedicines have drawbacks such as a complicated production process and a high cost. In order to realize high reproducibility, that is, stable supply and low cost accordingly, it is critical to establish sophisticated production techniques.
In general, proteins such as antibodies are produced by gene recombination instead of chemical protein synthesis. Such technique comprises inserting DNA encoding a certain protein into a vectore such as plasmid in an expressible or operable manner, culturing prokaryotic or eukaryotic cells transformed by the vector, and recovering and purifying the generated proteins.
As Shimizu et al. (Hiroshima University) have conducted studies on the gene amplification mechanism of cancer cells, they discovered that plasmids comprising the matrix attachment region (MAR)/mammalian replication initiation region (IR) efficiently caused gene amplification in cancer cells or other cells and that such plasmids were incorporated into the extrachromosomal double minute (DM) (Non-Patent Document 1 and Non-Patent Document 2). Using such DM, specifically, multimeric plasmids can be prepared in the transfected cells and stably partitioned to daughter cells. Thus, plasmids can be amplified at high levels in mammalian cells (Patent Document 1 and Non-Patent Document 3).
Shimizu et al. have proposed several methods for amplifying genes of interest in mammalian animal cells.
For example, Patent Document 2 discloses that a replication fork from the replication initiation region does not allow a poly (A) addition sequence or replication fork inhibitory sequence to be present in a region where the replication and transcription from an arbitrary gene integrated into a vector collide with each other, and Patent Document 2 also discloses a vector that allows such region to include a matrix attachment region.
Patent Document 3 and Patent Document 4 each disclose that a polynucleotide of 500 bp to 10,000 bp comprising a telomere repeat sequence is introduced as a new element into a mammalian animal cell simultaneously with the vector described above.
Patent Document 5 discloses that an IR/MAR-containing sequence forms DM with a single-stranded or double-stranded hairpin structure.
In addition, Shimizu et al. discloses Non-Patent Documents 4 to 6 related to the invention described above.
Non-Patent Document 7 describes as follows. When antibodies were produced using the IR/MAR plasmid, the antibody production level increased highly in the E. coli cell line COLO 320DM and the Chinese hamster ovary cell line CHO DG44. In the CHO K1 cell line, while the antibody production level was significantly low even when the plasmid comprised IR/MAR. Thus, efficient gene amplification was not observed.
With respect to the IR/MAR gene amplification technique described above, the documents mentioned above do not substantially disclose the use of the mammalian artificial chromosome vector developed by the present inventors (Patent Documents 6 to 9 and Non-Patent Documents 8 and 9).
As described in Patent Documents 3, 4, and the like, the presence of DM in addition to IR/MAR is necessary in order to amplify a DNA of interest (e.g., DNA encoding a protein). DM is an extrachromosomal circular DNA, and it comprises chromatin and is replicable, but it does not comprise a centromere or telomere. However, a gene amplified by IR/MAR has a problem that the expression of the gene is significantly lowered by silencing. In order to resolve the problem, it is essential to add a histone deacetylase (T-IDAC) inhibitor (e.g., butyrate) to a cell expressing the protein prepared by IR/MAR for removing the effect of silencing. This may adversely affect cell proliferation or damage cells. Thus, a protein production technique involving the use of IR/MAR is not suitable for use in a continuous culture system.
In order to resolve the problems described above, an object intended by the present inventors is to construct the IR/MAR system that does not comprise DM as an essential element, specifically to provide a method for high expression of a DNA sequence comprising: inserting IR/MAR and a DNA sequence of interest into a human artificial chromosome (HAC), or a mammalian artificial chromosome (MMAC) vector, suitable for stable gene expression comprising a mouse artificial chromosome (MAC) to prepare a novel IR/MAR-MMAC nucleic acid construct; and introducing the nucleic acid construct into a mammalian cell, thereby expressing the DNA sequence at a high level. Such technique has not yet been disclosed, and a possibility of gene amplification or stable protein production has not yet been known.
The present inventors conducted intensive studies in order to resolve the above-mentioned problems and, as a result, have now found that mammalian cells comprising a novel IR/MAR-MMAC nucleic acid construct would enable high expression of a DNA of interest.
Accordingly, the present invention includes the following features.
The present invention provides a method for gene amplification using a novel IR/MAR-MMAC nucleic acid construct. With respect to the use of such method for gene amplification, for example, mammalian cells transformed with the nucleic acid construct comprising DNA encoding a useful protein of interst may be cultured to enable stable and high expression of the protein of interest, or alternatively, when such DNA is a reporter gene, the expression of the gene may be elevated in cells, so that the change in the expression level is easily detected, leading to enhancing a detection sensitivity.
This description includes the content disclosed in Japanese Patent Application No. 2017-213237 (filed Nov. 2, 2017) from which the present application claims priority.
The present invention will be described in more detail.
The IR/MAR-MMAC nucleic acid construct of the present invention comprises elements, such as at least one DNA sequence of interest, a matrix attachment region (MAR), and a replication origin (also referred to as “replication initiation region” (IR)), in a mammalian artificial chromosome vector (MMAC) that is maintained independently from the host cell chromosome and is capable of stably expressing the DNA of interest, and such IR/MAR-MMAC nucleic acid construct is used for high expression of the DNA of interest. The vector comprising such elements is referred to as the “IR/MAR-MMAC nucleic acid construct” (e.g.,
Before the particular elements are inserted, the mammalian artificial chromosome (MMAC) vector comprises a chromosome fragment comprising the centromere derived from a chromosome of a certain mammal and the telomere bound to the terminus of the chromosome fragment.
The chromosome fragment may be prepared by telomere truncation described in, for example, WO 00/10383. Specifically, an artificial chromosome vector of interest may be obtained by homologous recombination using a targeting vector that carries an artificial telomere sequence and telomere truncation in a mammalian cell that carries a mammalian chromosome. In this case, a long arm region and a short arm region (if any) located near the centromere of the chromosome are cleaved by telomere truncation and the telomere is bound to the cleavage site. Also, it is preferable that the chromosome fragment substantially comprise no endogenous gene. Alternatively, at least 99.9% of the endogenous genes are preferably deleted from the chromosome fragment, and at least 99.99% of the endogenous genes are more preferably deleted therefrom.
The term “artificial telomere sequence” used herein refers to a telomere sequence that is artificially added by telomere truncation as described in, for example, WO 00/10383. For example, both the human telomere sequence and the mouse telomere sequence comprise a repeat sequence of TTAGGG.
Examples of mammals used herein include, but are not particularly limited to, primates, such as humans, monkeys, chimpanzees, and gorillas, rodents, such as mice, rats, hamsters, and guinea pigs, ungulates, such as bovines, sheep, and goats, dogs, and cats, preferably humans, mice, and rats.
The chromosome of any of the mammals may be preferably used for preparation of an artificial chromosome vector. It is preferable to select a chromosome that enables a mammalian cell to be transformed to stably accept an artificial chromosome vector. When rodents cells (e.g., CHO cell lines, such as K1, pro-3, DG44, DUKX-X11, or S), mouse myeloma cells (e.g., NSO or Sp2/0), or BHK cell lines (e.g., BHK21) are used as mammalian cells, for example, any of the rodent artificial chromosome vector (e.g., a mouse artificial chromosome (MAC) vector), an artificial chromosome vector derived from a hamster (e.g., an artificial chromosome vector derived from CHO), and a human artificial chromosome (HAC) vector may be introduced into rodent cells, and the mouse artificial chromosome vector may preferably be introduced thereinto. When a human cell line is used, in addition, it is preferable that the human artificial chromosome vector is introduced into a human cell line.
Concerning chromosomes, for example, mice have 40 chromosomes, rats have 42 chromosomes, and humans have 46 chromosomes. Any chromosomes may be used for preparation of a mammalian artificial chromosome (MMAC) vector. As a specific example of the use of such chromosome, the mammalian artificial chromosome (MMAC) vectors developed by the present inventors encompass the human artificial chromosome vector (JP Patent No. 4,895,100) and the mouse artificial chromosome vector (JP Patent No. 5,557,217 and JP Patent No. 4,997,544), and the chromosomes may be used to insert the elements, such as at least one DNA sequence of interest, a matrix attachment region (MAR), and a replication origin (i.e., a replication initiation region (IR)), into such vectors.
Specifically, the mammalian artificial chromosome (MMAC) vector may be prepared by the methods described in the patent documents exemplified above or the like. In addition, the vector comprises a DNA sequence insertion site into which foreign DNA is to be inserted. The DNA sequence insertion site is, for example, a site-directed recombinase recognition site. Examples of such systems include: a system of the Cre recombinase derived from bacteriophage P1 and the loxP sequence as a recognition site of the Cre recombinase (the Cre/loxP system); a system of the FLP enzyme derived from a budding yeast and the FRT sequence as a recognition site of the FLP enzyme (the Flp/FRT system); and a system of the φC31 integrase derived from Streptomyces phage and the attB/attP sequence as a recognition site of the integrase.
The mammalian artificial chromosome vector as used herein may be prepared by the top-down method that comprises performing mammalian chromosome-based chromosome modifications, or by the bottom-up method that comprises introducing a vector comprising a mammalian centromere sequence into mammalian cells (Yasuhiro Kazuki and Mitsuo Oshimura, The American Society of Gene & Cell Therapy 2011; doi:10.1038/mt.2011.136).
When preparing the IR/MAR-MMAC nucleic acid construct of the present invention, for example, a cassette comprising the DNA sequence of interest, the matrix attachment region (MAR), and the replication origin (i.e., the replication initiation region (IR)) may be inserted into a DNA sequence insertion site (e g , the loxP sequence) of the mammalian artificial chromosome (MMAC) vector. In general, the matrix attachment region (MAR) and the replication origin (i.e., the replication initiation region (IR)) are preferably linked in that order from the 5′ side, and such sequence is referred to as the “IR/MAR sequence” hereinbelow.
It is known that the IR/MAR sequence may be introduced in combination with a gene of interest into cells and may be used to amplify the copy number of the gene in the cells (e.g., Patent Documents 1 to 5). In the case of tumor cells, in addition, a plasmid comprising the IR/MAR sequence repeats multimerization to become a large cyclic molecule, and such large cyclic molecule is maintained in the form of a double minute (DM) in tumor cells. When DM is further integrated into the chromosome long arm of the nucleus in the tumor cell, it initiates the breakage-fusion-bridge (BFB) cycle to form a chromosomal homogeneously staining region (HSR), which serves as the gene amplification site in the tumor cell (Non-Patent Document 3). According to the conventional technique, however, continuous administration of an agent such as an HDAC inhibitor was necessary to keep the amplified gene to be expressed, and it was difficult to realize stable expression for a long period of time.
On the other hand, the IR/MAR-MMAC nucleic acid construct of the present invention is different from the DM or episome, although the mechanism is not elucidated, and, as a result, the construct is present on the the mammalian artificial chromosome (MMAC) vector in the state that foreign DNA is amplified in the nucleus, thus being clearly different from the DM in terms of its final form.
The IR/MAR-MMAC nucleic acid construct of the present invention comprises at least one DNA sequence of interest, the matrix attachment region (MAR), and the replication origin (i.e., the replication initiation region (IR)).
The matrix attachment region (MAR) comprises a polynucleotide derived from, for example, a matrix attachment region, such as mmunoglobulin light chain (Igκ) gene locus, Simian virus 40 (SV40) early region, or dihydrofolate reductase gene locus, although the region is not limited thereto (Tsutsui, K. et al., J. Biol. Chem.; 268: 12886-12894, 1993; Pommier, Y. et al., J. Virol.; 64:419-423, 1990; Shimizu, N. et al., Cancer Res.; 61: 6987-6990, 2001).
Examples of the replication origin (i.e., the replication initiation region (IR)) include, but are not limited to, replication initiation regions derived from the c-myc gene locus, the dihydrofolate reductase (DHFR) gene locus, and the β-globin gene locus (McWhinney, C. et al., Nucleic Acids Res.; 18: 1233-1242, 1990; Dijkwel, P. A. et al., Mol. Cell. Biol.; 8:5398-5409, 1988; Aladjem, M. et al., Science; 281:1005-1009, 1998).
Examples of DNA of interest include nucleic acids such as a gene or gene locus, cDNA, recombinant DNA, and modified DNA.
The term “protein” used herein refers to a protein, polypeptide, or peptide, unless otherwise noted. As long as it is industrially useful, the term refers to any protein, preferably a human-derived protein. Examples of such proteins include any proteins used for treatment, prevention, diagnosis, and the like, of diseases. Examples of such proteins include: antibody medicines, such as rheumatic arthritis-associated antibodies and cancer- or tumor-associated antibodies; therapeutic proteins, such as erythropoietin (EPO), G-CSF, GM-CSF, human growth hormone, blood coagulation proteins, platelet production-stimulating factor, and interferon; and interleukin, and other cytokines.
In the present invention, due to the use of the mammalian artificial chromosome (MMAC) vector, not only a gene,but also a gene locus can be inserted into the vector. When the protein to be expressed is an antibody protein, for example, the heavy chain gene locus (contained in chromosome 14 in case of human) and/or the light chain gene locus (contained in chromosome 2 (κ chain) and chromosome 22 (λ chain) in case of human), which comprise V region, D region, J region, and C region, may be used. When preparing a recombinant antibody, such as a single-stranded antibody comprising a heavy chain variable region (VH) ligated to a light chain variable region (VL) preferably via a linker, DNA encoding the antibody, may be used. In addition, in order to prepare therapeutic proteins as exemplified above, genes or cDNAs encoding the proteins and, optionally, modified DNAs may be used. The term “modified DNAs” used herein may include, for example, DNAs encoding a deletion, substitution, addition, or insertion of at least one amino acid of the proteins.
The IR/MAR-MMAC nucleic acid construct may further comprise one or two or more same or different types of elements, such as any of gene promoters, enhancers, poly A sequences, drug resistant genes, reporter genes (e.g., DNA encoding fluorescent proteins or luminescent proteins), DNA sequence insertion sites (e.g., loxP), and insulators. The elements may be inserted at an adequate site that is capable of causing the efficient amplification of a nucleic acid, such as DNA encoding a protein of interest. For example, the promoters and enhancers may be inserted at a given site in the 5′-untranslated region of the DNA. Examples of promoters include: viral promoters such as CMV, SV40, and CAG promoters; and non-viral promoters such as elongation factor (EF)-1 and mouse phosphoglycerate kinase (PGK) promoters.
The present invention further provides a mammalian cell for high expression of DNA of interest that comprises the IR/MAR-MMAC nucleic acid construct described in section 1 above.
Mammalian cells include, but not particularly limited to, Chinese hamster ovary (CHO) cells, tumor cells, COS cells, NIH3T3 cells, and BHK cells, as long as the nucleic acid amplification of DNA of interest occurs. Examples of CHO cells include CHO K1 cells (e.g., ATCC CCL-61, RIKEN RCB0285, and RIKEN RCB0403), CHO-oro-3 cells, CHO DG44 cells, and CHO DUKX-X11 cells. Examples of tumor cells include human colon cancer cells (e.g., COLO 320HSR cells such as ATCC CCL-220.1), HeLa cells, and mouse myeloma cell lines (e g , NSO cell and Sp2/0 cell). Mammalian cells preferable for gene amplification are CHO cells, more preferably CHO DG44 cells. Mammalian cells preferable for gene expression are CHO cells, more preferably CHO K1 cells.
In order to introduce a cassette comprising the DNA sequence of interest, the matrix attachment region (MAR), and the replication origin (i.e., the replication initiation region (IR)) into a desired mammalian cell, for example, the Cre-loxP system may be used. Specifically, Cre recombinase may be allowed to act on a cultured mammalian cell line (e.g., CHO cell line) comprising the mammalian artificial chromosome (MMAC) vector prepared in the manner as described in section 1 above, and then a cassette comprising the DNA sequence of interest, the matrix attachment region (MAR), and the replication origin (i.e., the replication initiation region (IR)) is inserted at the loxP site of MMAC to obtain the mammalian cell line comprising the IR/MAR-MMAC nucleic acid construct (e.g.,
As described in the examples below, in the present invention, the CHO K1 cell line is preferable as the mammalian cell line into which the IR/MAR-MMAC nucleic acid construct is introduced. As shown in, for example,
The present invention further provides a method for high expression of a protein encoded by DNA of interest comprising a step of culturing a mammalian cell comprising the IR/MAR-MMAC nucleic acid construct in a medium.
For mammalian cell culture, a medium suitable for the type of cells to be cultured is selected. Examples of basal medium that can be generally used include MEM medium, DMEM medium, RPMI medium, F12 medium, Macy's 5A medium, and a mixture thereof (e.g., DMEM/F12). The basal medium may further be supplemented with at least one component, such as serum, serum replacement, antibiotics, glutamic acid, or pyruvic acid. The medium has a pH of, for example, approximately 7.0 to 7.4.
Culture is carried out in the air containing CO2 at approximately 2% to 10% (approximately 5%, in general), and temperature of about 35° C. to 40° C. (about 37° C., in general) is suitable.
A culture period is generally 1 day to 3 months, preferably 1 day to 2 months.
Any culture technique may be selected from among, for example, batch culture, flask culture, fed-batch culture, continuous culture, suspension culture, shake culture, agitation culture, and circulation culture. In order to suppress cell aggregation, a culture solution may be allowed to, for example, pass through a hollow-fiber filter to disintegrate the aggregate during culture.
A culture apparatus used for animal cell culture may be used. The apparatus is preferably equipped with aeration, temperature control, agitation, pH adjustment, and DO adjustment functions. Culture may be carried out using, for example, a fermenter tank culture apparatus, an airlift culture apparatus, a fluidized bed culture apparatus, a hollow-fiber culture apparatus, a roller bottle culture apparatus, a packed bed culture apparatus, or a bag culture apparatus.
Another example of high expression of a protein includes high expression of a reporter gene used for monitoring the behavior or biological functions of a protein of interest in mammalian cells. For example, a reporter gene (e.g., DNA encoding a fluorescent protein or a luminescent protein) comprising a gene control sequence (e.g., a promoter or enhancer) ligated thereto may be operably inserted into DNA of interest. As a result of high expression of the reporter gene, detection sensitivity of reporter assays can be enhanced. This enables observation of the behavior of DNA of interest expressed by certain stimulation in cells using a florescence or luminescence as the indicator. Such observation may be generally carried out in laboratories, and cell culture may be thus performed using a solid medium in accordance with a cell type. In general, the observation may also be carried out using, for example, a fluorescent microscope with a camera.
The present invention further provides a method for producing a protein of interest comprising the following steps of: culturing mammalian cells comprising the IR/MAR-MMAC nucleic acid construct in a medium; and recovering (or collecting) the produced protein.
The step of culture is as described in (A.1) above.
In the step of cell recovery, for example, the supernatant or the cells after centrifugation, filtration, or other means may be subjected to column chromatography, such as affinity column chromatography, gel filtration chromatography, or ion exchange chromatography, filtration, ultrafiltration, salting out, dialysis, or a combination thereof, in order to obtain the protein produced by the culture. It is intended that the method of recovery is not limited to the methods described above and comprises any methods that can generally be employed for purification of proteins.
When the protein is particularly a glycoprotein such as an antibody, the glycoprotein may be carried out using a protein A column, a protein G column, a protein L column, or an IgG-binding peptide column.
The present invention further provides a non-human animal that carries the IR/MAR-MMAC nucleic acid construct.
A non-human animal may be prepared by, for example, introducing the nucleic acid construct into an ES cell or iPS cell, introducing the resulting cell into the blastocyst, transplanting the blastocyst nto the uterus of a surrogate mother, and allowing the surrogate mother to give birth to animals. The method of preparation is described below.
An ES cell may be established and maintained by removing an inner cell mass from the blastocyst of the fertilized egg of an animal of interest and by using the mitomycin C-treated mouse fetal fibroblast cell as a feeder (M. J. Evans and M. H. Kaufman, Nature, 292: 154-156, 1981).
The iPS cells generate a colony within approximately 3 to 5 weeks by introducing a certain reprogramming factor (DNA or a protein) into somatic cells (including somatic stem cells) and conducting culture and passage culture in an adequate medium. As reprogramming factors, for example, a combination of Oct3/4, Sox2, Klf4, and c-Myc, a combination of Oct3/4, Sox2, and Klf4, a combination of Oct4, Sox2, Nanog, and Lin28, and a combination of Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28 are known (K. Takahashi and S. Yamanaka, Cell 126: 663-676, 2006; WO 2007/069666; M. Nakagawa et al., Nat. Biotechnol., 26: 101-106, 2008; K. Takahashi et al., Cell 131: 861-872, 2007; J. Yu et al., Science 318: 1917-1920, 2007; and J. Liao et al., Cell Res., 18, 600-603 2008). For example, the mitomycin C-treated mouse fetal fibroblast cell line (e.g., STO) is used as the feeder cell, and vector-introduced somatic cells (approximately 104 to 105 cells/cm2) are cultured on the feeder cell layer using an ES cell culture medium at approximately 37° C. The feeder cell is not essential (Takahashi, K. et al., Cell 131: 861-872, 2007). Examples of basal media include Dulbecco's modified Eagle medium (DMEM), Ham's F-12 medium, and a mixture thereof. As an ES cell culture medium, for example, a mouse ES cell culture medium or a primate ES cell culture medium (ReproCELL Incorporated, Japan) may be used.
ES cells and iPS cells are known to contribute to the germ lines. Thus, such cells into which the IR/MAR-MMAC nucleic acid construct comprising the DNA sequence of interest has been introduced may be injected into the embryonic blastocyst of a mammal of the same species as with the origin of the cells, and the embryos are transplanted into the uterus of the surrogate mother to give birth to animals. Thus, non-human animals (or transgenic non-human animals) may be prepared. Further, the resulting female and male transgenic animals may be subjected to crossing to prepare homozygous animals and offspring animals thereof.
When a heterologous (e g , human) protein encoded by DNA of interest is to be prepared using the non-human animal, for example, an animal in which the endogenous gene equivalent to the DNA of interest has been deleted or completely inactivated may be used.
Examples of non-human animals include rodents, such as mice, rats, and hamsters.
The present invention will be described in more detail with reference to the following examples, although the technical scope of the present invention is not limited to these examples.
[Example 1] Establishment of CHO DG44 Cell Line Carrying HAC Vector Derived from Human Chromosome 21
[A] Introduction of HAC from CHO K1 Cell Comprising Human Chromosome 21-Derived HAC Vector into CHO DG44 Cell
In order to insert and amplify a gene of interest into/on the HAC in the CHO DG44 cell that exhibits high gene amplification efficiency achieved by the IR/MAR sequence (Y. Araki et al., Plos. One, 7.7. e41787, 2012), the HAC comprising the DNA sequence insertion site (loxP) is introduced into the CHO DG44 cell (
The used as the chromosomal donor cell was the CHO kkpqN cl.10 cell line derived from the CHO K1 cell that carries the HAC vector derived from human chromosome 21 (hereafter, referred to as “CHO kkpqN10,” JP 2007-295860 A). As the recipient cell, the CHO DG44 cell (provided by Dr. L.A. Chasin, Columbia University) was used. The CHO kkpqN10 cells were inoculated into twelve 25-cm2 centrifuge flasks (Nunc) and cultured in a medium (20% FBS, F12) containing colcemid (0.1 μg/ml, Gibco) for 72 hours to induce microcell formation. The centrifuge flasks were filled with a cytochalasin B (10 μg/ml in F12, Sigma) solution kept warm at 37° C. in advance, the centrifuge flasks were inserted into an acrylic centrifuge, and centrifugation was then carried out at 37° C. and 8,000 rpm (11,899×g) for 1 hour (JLA-10.5 rotor, Beckman). The microcells were suspended in the serum-free DMEM medium, recovered, and filtered and purified using SWINNEX-25 (Millipore) equipped with 8-μm, 5-μm, and 3-μm filters (Whatman), successively. The purified microcells were resuspended in 2 ml of F12 containing Phytohemagglutinin-P (Difco) at 50 μg/ml. The resuspended microcells were added in an amount of 1 ml each in two 6-cm dishes in which the CHO DG44 cells had been cultured to reach 90% saturation and then allowed to stand at 37° C. for 15 minutes. PEG1000 (final concentration: 50% (w/v), Sigma) and DMSO (final concentration: 7% (w/v), Sigma) were dissolved in DMEM, the solution filtered through a 0.22-μm filter (Corning) was applied to the cells over a period of 1 minute, and the cells were washed with serum-free DMEM. The cells were cultured in an F12 medium containing 10% FBS for 24 hours, dispersed by trypsin treatment, and then inoculated into six 10-cm dishes. The cells were cultured in a selection medium (10% FBS, F12) containing blasticidin (4 μg/ml) for 2 weeks, and drug-resistant colonies were then obtained. Microcell fusion was carried out twice to isolate 12 resistant colonies, which colonies were amplified and then subjected to the subsequent analysis (clone name: CHO DG44 N10).
From among the 12 CHO DG44 N10 cell clones obtained above, 4 clones were selected and subjected to FISH analysis using human Cot-1 DNA as a probe in accordance with the method described in the report by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175, 2001). It is possible to distinguish the CHO K1 cell-derived CHO kkpqN10 cell line from the CHO DG44 cell line on the basis of the karyotypic features, i.e. that, while the CHO K1 cell line comprises 3 large chromosomes, the CHO DG44 cell line comprises 2 large chromosomes (
From the results above, it was concluded that the human artificial chromosome (HAC) vector comprising the DNA sequence insertion site had been introduced into the CHO DG44 cell line.
[Example 2] Introduction of the 1R/MAR Sequence and the EGFP Gene into HAC
The loxP sequence to be integrated into HAC was amplified using the pPAC4 vector (obtained from BACPAC Resource Center, Children's Hospital Oakland Research Institute) as a template.
The primer oligonucleotide sequences prepared based on the nucleotide sequence obtained from the GenBank database (Accession Number: U75992.1) are shown below:
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-plus- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows: after thermal denaturation at 94° C. for 2 minutes, a cycle of 98° C. for 10 seconds and 68° C. for 50 seconds was conducted for 16 cycles. The PCR product was purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). Thereafter, the resultant was cleaved with the restriction enzyme StuI (NEB), electrophoresed on 1% agarose gel, cleaved from the gel, and then purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation).
The DNA fragment of the purified loxP sequence digested with the restriction enzyme StuI (NEB) and blunt-ended at both ends was cloned into the StuI site of the pCX-EGFP plasmid vector having the EGFP expression unit using Ligation high v2 (Toyobo Co., Ltd.) in accordance with the recommended protocol. A vector comprising the loxP sequence cloned thereinto in the inverse direction from the EGFP sequence was designated as the pCX-EGFP CMV loxP vector (
[A.1.2] Conversion of Drug Selection Marker in pCX-EGFP CMV loxP Vector
Because both the pCX-EGFP CMV loxP vector and the pΔBN AR1-Dhfr vector comprising the IR/MAR sequence integrated therein (provided by Dr. Noriaki Shimizu, Hiroshima University) comprise the ampicillin resistant gene (Amp), when the pCX-EGFP CMV loxP vector is inserted into the pΔBN AR1-Dhfr vector, then many self-ligating plasmid vectors are generated, but a plasmid comprising the pCX-EGFP CMV loxP vector sequence cloned into the pΔBN AR1-Dhfr vector might not be obtained. In order to improve efficiency when inserting the pCX-EGFP CMV loxP vector into pΔBN AR1-Dhfr comprising the IR/MAR sequence integrated therein, an experiment of converting the ampicillin resistant (Amp) gene of the pCX-EGFP CMV loxP vector into the kanamycin resistant (Km) gene was carried out. Here, details of the pΔBN AR1-Dhfr plasmid are described in C. Noguchi et al. Plos One. 7. 12. e52990. 2012.
The Km gene was amplified using the pPAC4 vector (described above) as a template. The primer oligonucleotide sequences prepared based on the nucleotide sequence obtained from the GenBank database (Accession Number: U75992.1) are shown below:
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-plus- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows: after thermal denaturation at 94° C. for 2 minutes, a cycle of 98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute and 10 seconds was conducted for 16 cycles. The PCR product was purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). Thereafter, the resultant was cleaved with the restriction enzyme ScaI/SpeI (NEB), electrophoresed on 1% agarose gel, cut out from the gel, and then purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). The DNA fragment of the purified Km gene digested with the restriction enzyme StuI (NEB) and blunt-ended at both ends was cloned into the Scal/Spel site of pCX-EGFP CMV loxP having the EGFP expression unit using Ligation high v2 (Toyobo Co., Ltd.) in accordance with the recommended protocol. A vector comprising the Km gene cloned thereinto was designated as the pCX-EGFP CMV loxP Km2 vector (
[A.2] Insertion of the EGFP loxP Sequence into the IR/MAR Vector
The pCX-EGFP CMV loxP Km2 vector was cleaved with the restriction enzyme PstI (NEB), electrophoresed on 1% agarose gel, cut out from the gel, and then purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). The end of the DNA fragment of the linearized pCX-EGFP CMV loxP Km2 vector was dephosphorylated using the rAPId Alkaline Phosphatase kit (Roche) in accordance with the recommended protocol in order to suppress self-ligation of the vector DNA fragment. The pΔBN AR1-Dhfr vector was also cleaved with the restriction enzyme PstI (NEB), electrophoresed on 1% agarose gel, cut out from the gel, and then purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). Subsequently, the dephosphorylated DNA fragment of the pCX-EGFP CMV loxP Km2 vector was ligated to the DNA fragment of the pΔBN AR1-Dhfr vector using Ligation high v2 (Toyobo Co., Ltd.) in accordance with the recommended protocol.
The plasmid vector to be integrated into HAC, which comprises the EGFP gene, the loxP sequence, and the IR/MAR sequence and was prepared by the steps described above, was designated as the pCX-EGFP loxP dhfr vector (
[A.3] Integration of the pCX-EGFP loxP dhfr Vector into HAC using the Cre/loxP System
Gene transfer was carried out using lipofection. In accordance with the protocol of Lipofectamine LTX (Invitrogen), 0.5 μg of the Cre-expressing pBS185 CMV-Cre vector (available from Addgene; Plasmid No. #11916) and 1 μg of the pCX-EGFP loxP dhfr vector were introduced into the CHO DG44 N10 cl.11 cells carrying the HAC vector for gene integration in 6 wells that had reached 90% confluency. Twenty four hours after transfection, the cells were inoculated into six 10-cm dishes each containing a selection medium consisting of F12 medium supplemented with 10% FBS and G418 300 μg/ml, and cultured in the medium for 2 weeks, thereby leading to appearance of resistant colonies, which contained many clones obtained by the single introduction. From among the obtained colonies, 24 clones were isolated, amplified, and then subjected to the subsequent analysis (clone name: CHO DG44 N10 MAR-HAC).
In order to prepare a control HAC that does not comprise the IR/MAR sequence, the gene transfer into the CHO DG44 N10 cl.11 cells was carried out in the same manner as that described above, except for the use of the pCX-EGFR loxP Km2 vector instead of the pCX-EGFP loxP dhfr vector. As a result, 3 clones were isolated, amplified, and then subjected to the experiment described below (clone name: CHO DG44 N10 EGFP-HAC).
In order to extract the genomic DNA of the G418-resistant cell line and select a recombinant as a template, PCR was carried out using the primers described below to confirm whether or not the EGFP and the IR/MAR sequence have been inserted into the human artificial chromosome (HAC) vector in a site-directed manner, in two junction regions appearing by recombination at the loxP region. The primer sequences are shown below.
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-FX- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows: after thermal denaturation at 95° C. for 10 minutes, a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds was conducted for 35 cycles. Out of 24 isolated CHO DG44 N10 MAR-HAC clones, 12 clones were analyzed and found to be positive. That is, the 12 clones were determined to comprise the EGFP and the IR/MAR sequence inserted into HAC and were subjected to the subsequent analysis (
In order to confirm amplification of EGFP gene based on integration of the IR/MAR sequence into HAC, the genome was extracted from the CHO DG44 N10 MAR-HAC clone, and comparative analysis was performed by real-time PCR using the CHO DG44 N10 EGFP-HAC clone comprising a single copy of the EGFP gene integrated therein (DG44 EGFP-MAC cl.12) as a control. (Note: the purpose of the PCR analysis was not to measure the absolute copy number of EGFP but was intended to conduct the experiments to simply determine whether or not the EGFP was amplified in comparison with the control.)
PCR was carried out using the StepOne real-time PCR System (Applied Biosystems) as a thermal cycler and, as Taq polymerase and a buffer, Power SYBR Green PCR master Mix (ThermoFisher) under the recommended conditions.
The primer sequences used are shown below. The EGFP primers and the NV1 primers as the genomic internal standards were prepared, respectively (Nissom PM et al., Biologicals, 35, 211-5, 2007).
From among the 24 CHO DG44 N10 MAR-HAC clones, 8 clones were analyzed by real-time PCR. As a result, all the analyzed clones were found to comprise a greater number of the EGFP sequences compared with the control CHO DG44 N10 EGFP-HAC. Among them, 3 clones comprising a particularly great number of EGFP sequences (cl.1, cl.2, and cl.3) were subjected to the subsequent analysis (
On the basis of the above results, the 3 clones were selected and subjected to two-color FISH analysis according to Matsubara et al. (FISH experimental protocol, Shujunsha Co., Ltd., Japan, 1994). FISH analysis was carried out using human cot-1 DNA and the pCX-EGFP plasmid as probes. As a result, all the clones were found to have larger chromosomes and a larger number of EGFP signals on human cot-1 signals, compared with the control (
[B.2] Analysis of EGFP expression level by Western blotting
In order to determine whether or not the EGFP expression of the CHO DG44 N10 MAR-HAC clone was increased accompanied with amplification of the gene sequence, the protein expression levels were analyzed by Western blotting.
Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with a pore size of 0.45 μm (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, the blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, then the reaction product was washed 4 times with PBS-T for 10 minutes and subjected to the subsequent antibody reaction. In the EGFP detection, the reaction was conducted using, as the first antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abcam: ab184601) for 1 hour at room temperature. After washed 3 times with PBS-T for 10 minutes, the subsequent reaction was conducted using, as the second antibody, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abcam: ab6789) for 45 minutes. After washed 3 times with PBS-T for 10 minutes, the further reaction was conducted using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan).
As the results of the Western blot analysis, higher expression levels were observed in the CHO DG44 N10 MAR-HAC clone, compared with the control (
[Example 3] Comparison of gene expression levels between the CHO K1 cell and the CHO DG44 cell
HAC comprising a single copy of EGFP was introduced into the CHO K1 cells and the CHO DG44 cells, and then the expression levels of EGFP from the same genome information were compared, whereby the protein production efficiency was compared between the cells.
[A] Integration of the EGFP Gene into HAC in the CHO kkpqN10 Cell Derived from the CHO K1 Cell
pCX-EGFP CMV loxP Km2 used in Example 1 is introduced into the CHO kkpqN10 cell, which is a CHO K1 cell carrying HAC for gene integration, so that a single copy of the EGFP gene is integrated into HAC via recombination using the Cre/loxP system.
Gene transfer was carried out via lipofection. In accordance with the protocol of Lipofectamine 2000 (Invitrogen), 5 μg of the Cre-expression vector pBS185 CMV-Cre (available from Addgene; Plasmid No. #11916) and 10 μg of the pCX-EGFP CMV loxP Km2 vector were introduced into the CHO kkpqN10 cells carrying the HAC vector for gene integration that had reached 90% confluency in 10-cm dishes (clone name: CHO KI NIO EGFP-HAC). Twenty four hours after transfection, the cells were inoculated into twenty 10-cm dishes each containing a selection medium comprising F12 medium supplemented with 10% FBS, Blasticidin S (Bsd) 8 μg/ml, and G418 800 μg/ml, followed by two-weeks culture, and, as a result, resistant colonies appeared, which contained many clones obtained by the single introduction, 42 clones of which were isolated, amplified, and then subjected to the subsequent analysis.
In order to select recombinants as templates following extraction of the genomic DNAs of Bsd- and G418-resistant cell lines, PCR was carried out using the primers indicated below, and whether or not the EGFP gene have been inserted into the human artificial chromosome (HAC) vector in a site-directed manner was confirmed at 2 junction regions that appeared by recombination in the loxP region. The primer sequences are as follows.
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-FX- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows: after thermal denaturation at 95° C. for 10 minutes, a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds was conducted for 35 cycles. Among the 42 isolated CHO K1 EGFP-HAC clones, 8 clones were found to be positive by analysis. The 8 clones were subjected to the subsequent analysis.
Among the 8 clones selected from the above results, 5 clones were subjected to two-color FISH analysis according to Matsubara et al. (FISH experimental protocols, Shujunsha Co., Ltd., 1994). FISH analysis was carried out using human cot-1 DNA and the pCX-EGFP plasmid as probes. As a result, a single clone (i.e., CHO K1 EGFP-HAC cl.27) was found to comprise the human artificial chromosome (HAC) at a retention rate of 100% and indicated EGFP-derived signals detected on HAC, thereby determining that the EGFP gene has been inserted into HAC in a site-directed manner (
[B] Transfer of EGFP-HAC from the CHO K1 N10 EGFP-HAC cl.27 Cell to the CHO DG44 Cell
As the chromosomal donor cell, the CHO K1 N10 EGFP-HAC cl.27 cell was used. As the recipient cell, the CHO DG44 cell (described above) was used. The CHO K1 N10 EGFP-HAC cl.27 cells were inoculated into twelve 25-cm2 centrifuge flasks (Nunc) and cultured in a medium (20% FBS, F12) containing colcemid (0.1 μg/ml, Gibco) for 72 hours to induce microcell formation. The centrifuge flasks were filled with a cytochalasin B (10 μg/ml in F12, Sigma) solution kept warm at 37° C. in advance, the centrifuge flasks were inserted into an acrylic centrifuge, and centrifugation was then carried out at 37° C. and 8,000 rpm (11,899×g) for 1 hour (JLA-10.5 rotor, Beckman). The microcells were suspended in the serum-free DMEM medium, recovered, and filtered and purified using SWINNEX-25 (Millipore) equipped with 8-μm, 5-μm, and 3-μm filters (Whatman), successively. The purified microcells were resuspended in 2 ml of F12 containing 50 μg/ml Phytohemagglutinin-P (Difco). The resuspended microcells were added in an amount of 1 ml each in two 6-cm dishes in which the CHO DG44 cells have been cultured to reach 90% saturation, and then allowed to stand at 37° C. for 15 minutes. PEG1000 (final concentration: 50% (w/v), Sigma) and DMSO (final concentration: 7% (w/v), Sigma) were dissolved in DMEM, the solution filtered through a 0.22-μm filter (Corning) was applied to the cells over a period of 1 minute, and the cells were washed with serum-free DMEM. The cells were cultured in an F12 medium containing 10% FBS for 24 hours, dispersed by trypsin treatment, and then inoculated into six 10-cm dishes. The cells were cultured in a selection medium (10% FBS, F12) containing blasticidin (4 μg/ml) for 2 weeks, and drug-resistant colonies were then obtained. Microcell fusion was carried out twice to isolate 14 resistant colonies, which were amplified and then subjected to the subsequent analysis (clone name: CHO DG44 EGFP-HAC).
Among the 14 clones selected from the above results, 3 clones were subjected to two-color FISH analysis according to Matsubara et al. (FISH experimental protocols, Shujunsha Co., Ltd., Japan, 1994). FISH analysis was carried out using human cot-1 DNA and the pCX-EGFP plasmid as probes. As a result, 2 clones (i.e., CHO DG44 EGFP-HAC cl.5 and cl.6) were found to have karyotypic features of the CHO DG44 cell, retain the human artificial chromosome (HAC) at a retention rate of 90% or higher, and show EGFP-derived signals detected therein. It was thus confirmed that EGFP-HAC had been transferred from the CHO K1 N10 EGFP-HAC cl.27 cell to the CHO DG44 cell (
[C] Comparative Analysis of the HAC-Derived EGFP Gene Expression Level between the CHO K1 Cell and the CHO DG44 Cell
As comparative cells, the CHO K1 N10 EGFP-HAC cl.27 cell derived from the CHO K1 cell line and the CHO DG44 EGFP-HAC cl.6 cell derived from the CHO DG44 cell were used. Since the HACs retained in the cells are of the same origin, it was considered possible to compare the EGFP gene expression levels in the intracellular environment.
Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with 0.45-μm pore size (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, a blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, the reaction product was washed 4 times with PBS-T for 10 minutes, and the resultant was subjected to the subsequent antibody reaction. EGFP detection was carried out by conducting a reaction using, as Antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abcam: ab184601) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using, as Antibody 2, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abcam: ab6789) for 45 minutes. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). EGFP protein quantification was carried out by measuring the band area using the Image J software and using the tubulin-corrected value.
As a result of the Western blot analysis, the protein expression level of the CHO K1 N10 EGFP-HAC clone was found to be 12.5 times higher than that of the CHO DG44 EGFP-HAC clone (
[Example 4] Transfer of the Gene Amplifying HAC Prepared using the MAR-HAC System into the Heterologous Cell
The EGFP gene amplified on HAC using the MAR-HAC in Example 2 was transferred into the CHO K1 cells on the basis of the finding of Example 3. Whether or not such transfer would further enhance the EGFP gene expression level was examined (
[A] Establishment of the Microcell-Mediated Chromosome Transfer Method using the CHO DG44 Cell
Since there was no achievement in the microcell-mediated chromosome transfer technique using the CHO DG44 cell in the past, the ability of the CHO DG44 cell for microcell formation was examined herein.
In order to confirm whether or not the CHO DG44 cell has the ability for microcell formation, colcemid treatment was carried out using the CHO DG44 N10 MAR-HAC cl.1 cell (described above). The CHO DG44 N10 MAR-HAC cl.1 cells were inoculated into 25-cm2 centrifuge flasks (Nunc) and cultured in a medium (20% FBS, F12) containing colcemid (0.1 μg/ml, Gibco) for 72 hours to induce microcell formation. As a result, formation of many microcells was confirmed (
From the results, the CHO DG44 cell was determined to have the ability for microcell formation, and the subsequent experiment for transfer of MAR-HAC into the CHO K1 cell via microcell-mediated chromosome transfer was carried out.
[B] Transfer of MAR-HAC into the CHO K1 Cell via Microcell-Mediated Chromosome Transfer
[B.1] Transfer of MAR-HAC into the CHO K1 Cell
As the chromosomal donor cell, the CHO DG44 N10 MAR-HAC cl.1 cell that carries HAC comprising the amplified EGFP gene integrated therein (described above) was used. As the recipient cell, the CHO kkpqN10 cell derived from the CHO K1 cell (described above) was used. The CHO DG44 N10 MAR-HAC cl.1 cells were inoculated into twelve 25-cm2 centrifuge flasks (Nunc) and cultured in a medium (20% FBS, F12) containing colcemid (0.1 μg/ml, Gibco) for 72 hours to induce microcell formation. The centrifuge flasks were filled with a cytochalasin B (10 μg/ml in F12, Sigma) solution kept warm at 37° C. in advance, the centrifuge flasks were inserted into an acrylic centrifuge, and centrifugation was then carried out at 37° C. and 8,000 rpm (11,899×g) for 1 hour (JLA-10.5 rotor, Beckman). The microcells were suspended in the serum-free DMEM medium, recovered, and filtered and purified using SWINNEX-25 (Millipore) equipped with 8-μm, 5-μm, and 3-μm filters (Whatman), successively. The purified microcells were resuspended in 2 ml of F12 containing 50 μg/ml Phytohemagglutinin-P (Difco). The resuspended microcells were added in an amount of 1 ml each in two 6-cm dishes in which the CHO kkpqN10 cells have been cultured to reach 90% saturation and then allowed to stand at 37° C. for 15 minutes. PEG1000 (final concentration: 50% (w/v), Sigma) and DMSO (final concentration: 7% (w/v), Sigma) were dissolved in DMEM, the solution filtered through a 0.22-μm filter (Corning) was applied to the cells over a period of 1 minute, and the cells were washed with serum-free DMEM. The cells were cultured in an F12 medium containing 10% FBS for 24 hours, dispersed by trypsin treatment, and then inoculated into six 10-cm dishes. The cells were cultured in a selection medium (10% FBS, F12) containing G418 (800 μg/ml) for 1 week, and drug-resistant colonies were then obtained. Microcell fusion was carried out 2 times to isolate 24 resistant colonies, and the isolated colonies were amplified and then subjected to the subsequent analysis (clone name: CHO K1R N10 MAR-HAC).
Among the 24 clones selected from the above results, 5 clones were subjected to two-color FISH analysis according to Matsubara et al. (FISH experimental protocols, Shujunsha Co., Ltd., Japan, 1994). FISH analysis was carried out using human cot-1 DNA and the pCX-EGFP plasmid as probes. As a result, all the clones were found to have karyotypic features of the CHO K1 cell and larger chromosomes stained with the human cot-1 probe, compared with common HAC. In addition, a plurality of EGFP-derived signals were detected thereon (
From the results above, transfer of MAR-HAC from the CHO DG44 MAR-HAC cl.1 cell to the CHO kkpqN10 cell was confirmed.
In order to confirm whether or not the EGFP expression levels would be enhanced via transfer of MAR-HAC into the CHO K1 cell, the EGFP expression levels were compared between the CHO DG44 MAR-HAC cl.1 cell and the CHO K1R N10 MAR-HAC cell clone.
Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with 0.45-μm pore size (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, a blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, the reaction product was washed 4 times with PBS-T for 10 minutes, and the resultant was subjected to the subsequent antibody reaction. EGFP detection was carried out by conducting a reaction using, as Antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abcam: ab184601) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using, as Antibody 2, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abeam: ab6789) for 45 minutes. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). EGFP protein quantification was carried out by measuring the band area using the Image J software and using the tubulin-corrected value.
As a result of the Western blot analysis, the protein expression level of the CHO K1R N10 MAR-HAC cell clone was found to be 2.01 to 2.85 times higher than that of the donor CHO DG44 MAR-HAC cl.1 cell (
[C.2] Comparison of EGFP Expression Level between HAC Comprising a Single Copy of the EGFP Gene and MAR-HAC
In order to accurately examine the effects of gene amplification using the MAR-HAC system, EGFP expression levels were compared between HAC comprising a single copy of the EGFP gene and HAC comprising EGFP amplified using the MAR-HAC system by conducting experiments using, as a host, the CHO K1 cell with high protein expression efficiency.
As the cell comprising the HAC in which a single copy of the EGFP gene was integrated, the CHO K1 N10 EGFP-HAC cl.27 cell (described above) was used. As the cell comprising HAC comprising EGFP amplified using the MAR-HAC system, the CHO K1R N10 MAR-HAC cell clone was used.
Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with a pore size of 0.45 μm (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, a blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, the reaction product was washed 4 times with PBS-T for 10 minutes, and the resultant was subjected to the subsequent antibody reaction. EGFP detection was carried out by conducting a reaction using, as Antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abcam: ab184601) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using, as Antibody 2, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abcam: ab6789) for 45 minutes. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFTLM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). EGFP protein quantification was carried out by measuring the band area using the Image J software and using the tubulin-corrected value.
As a result of the Western blot analysis, the protein expression level of the CHO K1R N10 MAR-HAC cell clone was found to be 17.3 to 25.7 times higher than that of the CHO K1 N10 EGFP-HAC cl.27 cell (
In order to examine whether or not the amplified gene on HAC constructed using the MAR-HAC system can be stably expressed for a long period of time, long-term passage cultures were carried out using the CHO K1R N10 MAR-HAC cl.13 cell (described above), and the EGFP expression level of the primary culture and those of the passage cultures were compared. The culture periods were determined by measuring the population doubling levels (PDL), and the EGFP expression levels were compared and analyzed by Western blotting at the stages of PDL3, PDL30, and PDL50.
Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with a pore size of 0.45 μm (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, a blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, the reaction product was washed 4 times with PBS-T for 10 minutes, and the resultant was subjected to the subsequent antibody reaction. EGFP detection was carried out by conducting a reaction using, as Antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abeam: ab184601) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using, as Antibody 2, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abcam: ab6789) for 45 minutes. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). EGFP protein quantification was carried out by measuring the band area using the Image J software and using the tubulin-corrected value.
As a result of the Western blot analysis, the expression level at PDL30 was found to be as lower as 62% when compared with the expression level at PDL3, while the expression level was maintain at 80% in case of PDL50 (
[Example 6] Amplification and Production of the Anti-VEGF Antibody Gene using the MAR-HAC System
The enhancer sequence A7 having a function of enhancing gene expression was cloned, the heavy chain gene and the light chain gene of the anti-VEGF antibody were prepared as artificially synthesized genes, and the anti-VEGF antibody-producing vector comprising the A7 element was prepared. The method of preparation will be described below in detail.
In order to prepare for a subcloning vector, the inspB4ins2 vector (provided by Dr. Tetsuya Ohbayashi, Research Initiative Center, Organization for Research Initiation and Promotion, Tottori University, Japan) was digested with AvrII (NEB) and Spel-HF (NEB), and the resultant was electrophoresed on 0.8% agarose gel, followed by purification with the MonoFas DNA purification kit (GL Sciences). The purified fragments were subjected to self-ligation using Ligation High ver.2 (Toyobo Co., Ltd.) to prepare the pB4ins1 subcloning vector. The inspB4ins2 vector is described in detail in Y. Yoshimura et al., Transgenic Research, 24. 4. pp. 717-727, 2015.
The A7 region is a 8,420-bp polynucleotide located in the position from88992123 to 89000542 of human chromosome 11 (Accession Number: AP003400.2). On the basis of the sequence information, the following primers were designed.
Using the primers prepared, PCR was carried out under the conditions described below using the genomic DNA of human IMR-90 cell (obtained from ATCC) as a template and KOD Fx DNA polymerase (Toyobo Co., Ltd.); i.e., after pre-denaturation at 95° C. for 2 minutes, a cycle of 98° C. for 10 seconds and 68° C. for 10 minutes was conducted for 35 cycles. The PCR product was purified using the MonoFas DNA purification kit. The purified product was digested with MluI (NEB) and then inserted into a vector prepared by digesting pB4ins1 with NruI (NEB) and MluI using Ligation High ver.2 (Toyobo Co., Ltd.), resulting in production of pB4-A7.
[A.1.3] Synthesis of the Genes that Produce the VEGF Antibody
Concerning the anti-VEGF antibody gene, the amino acid sequences of the heavy chain gene and the light chain gene were obtained from the GenBank database (Accession Numbers: KX119517 and KX119516). In addition, as a promoter for gene expression, the promoter sequence of the human RPS7 gene was obtained based on the nucleotide sequence obtained from the GenBank database (Accession Number: NG 011744.1). On the basis of these sequence information, the preparation of synthetic genes was consigned to FASMAC, and the following plasmid vectors comprising the anti-VEGF antibody gene were prepared (
The synthesized light chain vector, pUCFa-RPS7-VEGF-Lc, was digested with the restriction enzymes AscI (NEB) and XhoI (NEB), and the synthesized heavy chain vector, pUCFa-RPS7-VEGF-Hc, was digested with the restriction enzymes MluI-HF (NEB) and XhoI, followed by agarose gel electrophoresis. The fragments of the intended sizes were purified using the MonoFas DNA purification kit, and the VEGF light chain and the VEGF heavy chain were ligated using Ligation High ver.2 (Toyobo Co., Ltd.) to prepare the pUC-Fa-RPS7-VEGF-LcHc vector.
pB4-A7 was digested with NaeI (NEB) and MluI, and pUC-Fa-RPS7-VEGF-LcHc was digested with MluI and SnaBI (NEB), followed by agarose gel electrophoresis. The fragments of the intended sizes were purified using the MonoFas DNA purification kit, and the fragments were ligated using Ligation High ver.2 (Toyobo Co., Ltd.) to prepare the pUC-Fa-A7-RPS7-VEGF-LcHc vector comprising the A7 element.
[A.1.5] Introduction of Multiple Cloning Sites into IR/MAR Vector
In order to insert the antibody gene into the IR/MAR vector, additional restriction enzyme sites; i.e., PacI, AscI, FseI, and SacII, and a IoxG sequence for recombination were inserted into the PstI site of pΔBN AR1-Dhfr to construct a vector. The method for the preparation will be described below.
The pCX-EGFP CMV loxG Km vector used in Example 2 was used as a template to clone a sequence comprising the loxP sequence and then to introduce a plurality of restriction enzyme sites via PCR. The primer sequences are shown below.
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-FX- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows; i.e., after thermal denaturation at 94° C. for 2 minutes, a cycle of 98° C. for 10 seconds and 68° C. for 3 minutes was conducted for 20 cycles. The PCR product was purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). Thereafter, the purified product was cleaved with the restriction enzyme PstI (NEB), electrophoresed on 1% agarose gel, cut out from the gel, and then purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). Subsequently, this PCR product was introduced at the PstI (NEB) site of the pΔBN AR1-Dhfr vector in the manner described below. The pΔBN AR1-Dhfr vector was cleaved with the restriction enzyme PstI (NEB), electrophoresed on 1% agarose gel, cut out from the gel, and then purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). In order to prevent self-ligation of the DNA fragment of the linearized pΔBN AR1-Dhfr vector, the ends of the DNA fragment were dephosphorylated using the rAPId Alkaline Phosphatase kit (Roche) in accordance with the recommended protocol. Subsequently, the DNA fragment comprising loxP derived from the pCX-EGFP CMV loxG Km vector was ligated to the dephosphorylated DNA fragment of the pΔBN AR1-Dhfr vector using Ligation high v2 (Toyobo Co., Ltd.) in accordance with the recommended protocol. The final form of the modified 1R/MAR vector prepared by inserting additional restriction enzyme sites (PacI, AscI, FseI, and SacII) and the loxG sequence into pΔBN AR1-Dhfr was designated as the “pΔBN AR1-Dhfr loxG Km2 MSC vector” (
[A.1.6] Introduction of the Anti-VEGF Antibody Gene into the IR/MAR Vector
The IR/MAR vector (pΔBN AR1-Dhfr loxG Km2 MSC) was digested with AscI and dephosphorylated using Antarctic Phosphatase (NEB). The pUC-Fa-A7-RPS7-VEGF-LcHc was digested with AscI and separated via agarose gel electrophoresis. These DNA fragments were purified using the MonoFas DNA purification kit and ligated using Ligation High ver.2. The thus obtained anti-VEGF antibody-producing IR/MAR vector for artificial chromosome integration was designated as “A7-VEGF IR MAR” (
[A.2] Integration of the Anti-VEGF Antibody Gene Vector into HAC using the Cre/loxP System
Gene transfer was carried out via lipofection. In accordance with the protocol of Lipofectamine LTX (Invitrogen), 0.5 μg of the Cre-expression vector pBS185 CMV-Cre (available from Addgene; Plasmid No. #11916) and 1 μg of the A7-VEGF IR MAR vector were introduced into the CHO DG44 N10 cl.11 cells carrying the HAC vector for gene transfer in 6 wells, which cells have reached 90% confluency. Twenty four hours after transfection, the cells were inoculated into six 10-cm dishes each containing a selection medium comprising F12 medium supplemented with 10% FBS and G418 300 μg/ml, followed by 2-weeks culture, and, as a result, resistant colonies appeared, 18 clones of which were isolated, amplified, and then subjected to the subsequent analysis (clone name: CHO DG44 VEGF MAR-HAC).
In order to prepare a control HAC that does not comprise the IR/MAR sequence, the gene transfer into the CHO DG44 N10 cl.11 cells was carried out in the manner described above, using the A7-VEGF loxG vector. As a result, 22 clones were isolated, amplified, and then subjected to the experiment described below (clone name: CHO DG44 VEGF-HAC).
In order to extract the genomic DNA of a G418-resistant cell line and to select recombinants as templates, PCR was carried out using the primers described below to confirm whether or not the EGFP and the IR/MAR sequence have been inserted into the human artificial chromosome (HAC) vector in a site-directed manner, in two junction regions appearing by recombination at the loxP region. The primer sequences are shown below.
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-FX- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows; i e , after thermal denaturation at 95° C. for 10 minutes, a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds was conducted for 35 cycles. Among the 18 isolated CHO DG44 VEGF MAR-HAC clones, 9 clones were analyzed and found to be positive. That is, the 9 clones were evaluated to comprise the anti-VEGF antibody gene and the IR/MAR sequence inserted into HAC, and they were then subjected to the subsequent analysis (
In order to confirm that the anti-VEGF antibody gene was amplified due to the integration of the IR/MAR sequence into HAC, the genome was extracted from the CHO DG44 VEGF MAR-HAC clone, and comparative analysis was performed via real-time PCR using, as a control, the CHO DG44 VEGF-HAC clone comprising a single copy of the anti-VEGF antibody gene integrated therein. (Note: the PCR analysis was not intended to measure the absolute copy number of the anti-VEGF antibody genes but was intended to conduct the experiment to simply measure the gene amplification as compared with the control.)
PCR was carried out using the StepOne real-time PCR System (Applied Biosystems) as a thermal cycler, and Power SYBR Green PCR master Mix (ThermoFisher) as Taq polymerase and a buffer, under the recommended conditions.
The primer sequences used are shown below. The primers for the anti-VEGF antibody gene heavy chain (Hc) and light chain (Lc) and the NV1 primers for the internal genome standard were used (Nissom PM et al., Biologicals, 35, 211-5, 2007).
Among the 9 CHO DG44 VEGF MAR-HAC clones, 5 clones were analyzed by real-time PCR. As a result, 4 clones were found to comprise anti-VEGF antibody gene sequences more than the control cell comprising a single copy of the anti-VEGF antibody gene (CHO DG44 VEGF-HAC cl.7). In particular, the 4 clones comprising many anti-VEGF antibody gene sequences (i.e., cl.8, cl.9, cl.10, and cl.13) were subjected to the subsequent analysis (
For the 4 clones selected from the above-mentioned results, two-color FISH analysis was conducted according to Matsubara et al. (FISH experimental protocols, Shujunsha Co., Ltd., Japan, 1994). The FISH analysis was carried out using human cot-1 DNA and the A7-VEGF-loxG plasmid vector as probes. As a result, a single clone (i.e., CHO DG44 VEGF MAR-HAC cl.8) was found to maintaine independently from the host chromosome in the cell, and the clone had larger chromosomes and a larger number of anti-VEGF antibody gene signals on human cot-1 signals, when compared with the control (CHO DG44 VEGF-HAC cl.7) (
[B.5] Analysis of Anti-VEGF Antibody Production Level via Antibody Purification from Culture Supernatant
In order to examine whether or not the expression level of the anti-VEGF antibody of the CHO DG44 VEGF MAR-HAC clone was increased accompanied with gene sequence amplification, the antibodies produced from the culture supernatants were purified, and the concentrations thereof were measured and analyzed. Antibody purification was carried out using the spin-column-based IgG antibody purification kit (APK-10G, Cosmo Bio Co., Ltd., Japan) in accordance with the instructions included in the kit. Antibody concentration was measured using an absorption spectrometer (Scrum Inc.; Denovix). As a control, CHO DG44 VEGF-HAC cl.7 was used, and CHO DG44 VEGF MAR-HAC cl.8 was used as a cell that carries IR/MAR. The DG44 culture supernatant was obtained by seeding each of the cells in a 6-cm culture dish at cell density of 1 x 105 cells/ml, 3 times every 2 days over a period of 6 days, followed by purifying the antibody from the culture supernatant and then measuring the concentration of the antibody. As a result, although the growth rate was found to be substantially the same between the cells (
In order to confirm whether or not the antibody produced from each of the DG44 cells could recognize the VEGF protein being a specific antigen, enzyme linked immunosorbent assay (ELISA) was carried out. ELISA was carried out by adding 100 μ1 each of the VEGF antigen protein (Sino Biological: 11086-HNAH) diluted at 50 ng/well with PBS to the 96-well plate onto which the protein can be immobilized (442404, Thermo Fisher), and allowing the plate to stand at room temperature for 1 hour to immobilize the antigen. Subsequently, the plate was washed 3 times with TBS-T (Wako), 350 μ1 of 5% skim milk/T-BST (Wako) was added as a blocking buffer, and the plate was then allowed to stand at room temperature for 1 hour. The plate was washed 3 times with TBS-T (Wako), 100 each of the DG44 cell culture supernatant was used as a primary antigen, and the plate was then allowed to stand at room temperature for 1 hour. Thereafter, the plate was washed 3 times with TBS-T (Wako), 100 μl each of the antibody reacting with the human antibody (goat anti-human IgG HRP, BETHYL, A80-119P) diluted to 1/50000-fold was added as a secondary antibody, and the plate was then allowed to stand at room temperature for 30 minutes. The plate was washed 3 times with TBS-T (Wako), OPD, an enzyme substrate, was added at 0.5 mg/ml to a substrate buffer (Thermo Fisher), a solution containing H2O2 (Wako) at an amount of 1/1000 was added in an amount of 1,000 μl each, and the reaction was allowed to proceed for 30 minutes. Thereafter, 1 M H2SO4(Wako) was added to terminate the reaction, the wavelength at A492 nm was measured using an absorption plate reader (EPOCH2, Baio Tek), and the antibody reaction was then quantified.
As a result, it was confirmed that antibodies specifically reacting with the VEGF antigen were produced from the cells in correlation with the results of measurement of antibody concentration (
[Example 7] Establishment of CHO DG44 Cell Line Carrying MAC Vector Derived from Mouse Chromosome 11
[A] Introduction of HAC from CHO K1 Cell Comprising Mouse Chromosome 11-Derived MAC Vector into CHO DG44 Cell
Application of the gene amplification system to the mouse artificial chromosome (MAC) in addition to the human artificial chromosome was examined. The MAC vector is described in detail in JP 2011-549044 A. In order to insert and amplify a gene of interest into/on the MAC in the CHO DG44 cell that exhibits high gene amplification efficiency achieved by the IR/MAR sequence, MAC comprising the DNA sequence insertion site (loxP) is introduced into the CHO DG44 cell (
As the chromosomal donor cell, the CHO K1 MAC cell line derived from the CHO K1 cell that carries the MAC vector derived from mouse chromosome 11 (hereafter, referred to as “CHO K1 MAC,” JP 2011-549044 A) was used. As the recipient cell, the CHO DG44 cell (provided by Dr. L.A. Chasin, Columbia University) was used. The CHO K1 MAC cells were inoculated into twelve 25-cm2 centrifuge flasks (Nunc) and cultured in a medium (20% FBS, F12) containing colcemid (0.1 μg/ml, Gibco) for 72 hours to induce microcell formation.
The centrifuge flasks were filled with a cytochalasin B (10 μg/ml in F12, Sigma) solution kept warm at 37° C. in advance, the centrifuge flasks were inserted into an acrylic centrifuge, and centrifugation was then carried out at 37° C. and 8,000 rpm (11,899×g) for 1 hour (JLA-10.5 rotor, Beckman). The microcells were suspended in the serum-free DMEM medium, recovered, and filtered and purified using SWINNEX-25 (Millipore) equipped with 8-μm, 5-μm, and 3-μm filters (Whatman), successively. The purified microcells were resuspended in 2 ml of F12 containing Phytohemagglutinin-P (Difco) at 50 μg/ml. The resuspended microcells were added in an amount of 1 ml each in two 6-cm dishes in which the CHO DG44 cells have been cultured to reach 90% saturation, and then were allowed to stand at 37° C. for 15 minutes. PEG1000 (final concentration: 50% (w/v), Sigma) and DMSO (final concentration: 7% (w/v), Sigma) were dissolved in DMEM, the solution filtered through a 0.22-μm filter (Corning) was applied to the cells over a period of 1 minute, and the cells were washed with serum-free DMEM. The cells were cultured in an F12 medium containing 10% FBS for 24 hours, dispersed by trypsin treatment, and then inoculated into six 10-cm dishes. The cells were cultured in a selection medium (10% FBS, F12) containing blasticidin (4 μg/ml) for 2 weeks, and drug-resistant colonies were then obtained. Microcell fusion was carried out to isolate 4 resistant colonies, which were then amplified for the subsequent analysis (clone name: CHO DG44 MAC).
In accordance with the method described in the report by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175, 2001), the 4 CHO DG44 MAC cell clones obtained above were subjected to FISH analysis using mouse Cot-1 DNA as a probe. It is possible to distinguish the CHO K1 cell-derived CHO K1 MAC from the CHO DG44 cell line based on the karyotypic features. While the CHO K1 cell line comprises 3 large chromosomes, the CHO DG44 cell line comprises 2 large chromosomes (
From the above-mentioned results, it was concluded that the mouse artificial chromosome (MAC) vector comprising the DNA sequence insertion site was introduced into the CHO DG44 cell line.
[Example 8] Introduction of the IR/MAR Sequence and the EGFP Gene into MAC
Gene transfer was carried out via lipofection. In accordance with the protocol of Lipofectamine LTX (Invitrogen), 0.5 μg of the Cre-expression vector pBS185 CMV-Cre (available from Addgene; Plasmid No. #11916) and 1 μg of the pCX-EGFP loxP dhfr vector were introduced into the CHO DG44 MAC cl.611-1 and cl.611-4 cells, which have reached 90% confluency, carrying the MAC vector for gene integration in 6 wells. Twenty four hours after transfection, the cells were inoculated into six 10-cm dishes each containing a selection medium that comprises a F12 medium supplemented with 10% FBS and 300 μg/ml G418, and then culture was conducted therein for 2 weeks, whereby resistant colonies appeared, which contained many clones obtained by the single introduction. Among them, 6 clones were isolated, amplified, and then subjected to the subsequent analysis (clone name: CHO DG44 MAR-MAC).
In order to prepare a control MAC that does not comprise the IR/MAR sequence, the gene transfer into the cl.611-1 and cl.611-4 cells was carried out in the same manner as described above, using the pCX-EGFR loxP Km2 vector instead of the pCX-EGFP loxP dhfr vector. As a result, 6 clones were isolated, amplified, and then subjected to the experiments described below (clone name: CHO DG44 EGFP-MAC).
In order to select recombinants, the genomic DNA of the G418-resistant cell line was extracted, then PCR was carried out using the extracted genomic DNA, as templates, and the primers indicated below, and whether or not the EGFP and the IR/MAR sequence have been inserted into the mouse artificial chromosome (MAC) vector in a site-directed manner was confirmed at an junction region that appeared by recombination in the loxP region The primer sequences are as follows.
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-FX- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows; i.e., after thermal denaturation at 95° C. for 10 minutes, a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds was conducted for 35 cycles. The 6 isolated CHO DG44 MAR-MAC clones were analyzed and found to be positive. That is, the 6 clones were evaluated to comprise the EGFP and the IR/MAR sequence inserted into MAC, and were then subjected to the subsequent analysis (
In order to confirm that the EGFP gene was amplificed due to the integration of the IR/MAR sequence into MAC, the genome was extracted from the CHO DG44 MAR-MAC clone, and comparative analysis was performed by real-time PCR using, as a control, the CHO DG44 EGFP-MAC clone with integrated single copy of the EGFP gene. (Note: the PCR analysis was not intended to measure the absolute copy number of EGFP but was intended to conduct the experiments to simply determine whether or not the EGFP was amplified in comparison with the control.
PCR was carried out using the StepOne real-time PCR System (Applied Biosystems) as a thermal cycler and Power SYBR Green PCR master Mix (ThermoFisher) as Taq polymerase and a buffer, under the recommended conditions.
The primer sequences used are shown below. The EGFP primers and the NV1 primers as the genomic internal standards were prepared, respectively (Nissom PM et al., Biologicals, 35, 211-5, 2007).
The 6 CHO DG44 MAR-MAC clones obtained were analyzed by real-time PCR. As a result, a single clone was found to comprise EGFP sequences more than the control CHO DG44 EGFP-MAC cl.12. This clone was subjected to the subsequent analysis (
For 3 clones selected from the above-mentioned results, two-color FISH analysis was conducted according to Matsubara et al. (FISH experimental protocols, Shujunsha Co., Ltd., Japan, 1994). The FISH analysis was carried out using mouse cot-1 DNA and pCX-EGFP plasmid as probes. As a result, a single clone was found to maintaine the EGFP gene independently from the host chromosome ,and the clone had larger chromosomes and many EGFP signals on mouse cot-1 signals, when compared with the control (DG44 EGFP-MAC cl.12) (
In order to confirm whether or not the EGFP expression level of the CHO DG44 MAR-MAC clone was increased accompanied with gene sequence amplification, the protein expression levels were analyzed by Western blotting.
The Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with a pore size of 0.45 μm (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, a blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, the reaction product was washed 4 times with PBS-T for 10 minutes, and the resultant was subjected to the subsequent antibody reaction. EGFP detection was carried out by conducting a reaction using, as Antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abcam: ab184601) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using, as Antibody 2, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abcam: ab6789) for 45 minutes. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan).
As a result of Western blot analysis, higher protein expression levels were observed in the CHO DG44 MAR-MA clones, when compared with the control (
[Example 9] Establishment of the CHO DG44 Cell Line Carrying the Bottom-Up Type of Artificial Chromosome (tet-O HAC)
[A] Introduction of HAC from the CHO K1 Cell Comprising the Bottom-Up Type of Artificial Chromosome (tet-O HAC) Vector into the CHO DG44 Cell
In this Example, whether or not the gene amplification system is applicable to the bottom-up type of an artificial chromosome in addition to the top-down type pf an artificial chromosome was examined. The bottom-up type of artificial chromosome tet-O HAC vector is described in detail in Y. Iida et al., ACS Synth. Biol., 21. 3. pp. 83-90, 2014. In order to insert and amplify a gene of interest into/on the HAC in the CHO DG44 cell that exhibits high gene amplification efficiency achieved by the IR/MAR sequence, the tet-O HAC comprising the DNA sequence insertion site (loxP) is introduced into the CHO DG44 cell (
As the chromosomal donor cell, the CHO K1 tet-O HAC cell line derived from the CHO K1 cell that carries the tet-O HAC vector was used. As the recipient cell, the CHO DG44 cell (provided by Dr. L. A. Chasin, Columbia University) was used. The CHO kkpqN10 cells were inoculated into twelve 25-cm2 centrifuge flasks (Nunc) and cultured in a medium (20% FBS, F12) containing colcemid (0.1 μg/ml, Gibco) for 72 hours to induce microcell formation. The centrifuge flasks were filled with a cytochalasin B (10 μg/ml in F12, Sigma) solution kept warm at 37° C. in advance, the centrifuge flasks were inserted into an acrylic centrifuge, and centrifugation was then carried out at 37° C. and 8,000 rpm (11,899×g) for 1 hour (JLA-10.5 rotor, Beckman). The microcells were suspended in the serum-free DMEM medium, recovered, and filtered and purified using SWINNEX-25 (Millipore) equipped with 8-μm, 5-μm, and 3-μm filters (Whatman), successively. The purified microcells were resuspended in 2 ml of F12 containing Phytohemagglutinin-P (Difco) at 50 μg/ml. The resuspended microcells were added in an amount of 1 ml each in two 6-cm dishes in which the CHO DG44 cells have been cultured to reach 90% saturation, and then were allowed to stand at 37° C. for 15 minutes. PEG1000 (final concentration: 50% (w/v), Sigma) and DMSO (final concentration: 7% (w/v), Sigma) were dissolved in DMEM, the solution filtered through a 0.22-μm filter (Corning) was applied to the cells over a period of 1 minute, and the cells were washed with serum-free DMEM. The cells were cultured in an F12 medium containing 10% FBS for 24 hours, dispersed by trypsin treatment, and then inoculated into six 10-cm dishes. The cells were cultured in a selection medium (10% FBS, F12) containing blasticidin (4 μg/ml) for 2 weeks, and drug-resistant colonies were then obtained. Since many resistant colonies were obtained as a result of microcell fusion, 12 clones were isolated, amplified, and then subjected to the subsequent analysis (clone name: CHO DG44 tet-O HAC).
In accordance with the method described in the report by Shinohara et al. (Human Molecular Genetics, 10: 1163-1175, 2001), 9 clones selected from among the 12 CHO DG44 tet-O HAC cell clones obtained above were subjected to FISH analysis using human Cot-1 DNA as a probe. It is possible to distinguish the CHO K1 cell-derived CHO K1 tet-O HAC from the CHO DG44 cell line based on the karyotypic features. While the CHO K1 cell line comprises 3 large chromosomes, the CHO DG44 cell line comprises 2 large chromosomes (
From the above-mentioned results, it was concluded that the bottom-up type of artificial chromosome tet-O HAC vector, which comprises the DNA sequence insertion site, have been introduced into the CHO DG44 cell line.
[Example 10] Introduction of the 1R/MAR Sequence and the EGFP Gene into the tet-O HAC Vector
Gene transfer was carried out by lipofection. In accordance with the protocol of Lipofectamine LTX (Invitrogen), 0.5 μg of the Cre-expressing pBS185 CMV-Cre vector (available from Addgene; Plasmid No. #11916) and 1 μg of the pCX-EGFP loxP dhfr vector were introduced into the CHO DG44 tet-O HAC cl.6 and cl.9 cells, in 6 wells, that have reached 90% confluency. Twenty four hours after transfection, the cells were inoculated into six 10-cm dishes each containing a selection medium that comprises an F12 medium supplemented with 10% FBS and 300 μg/ml G418, and then the culture was conducted therein for 2 weeks, whereby resistant colonies appeared, which contained many clones obtained by the single introduction. Among them, 16 clones were isolated, amplified, and then subjected to the subsequent analysis (clone name: CHO DG44 MAR-tet-O HAC).
In order to select recombinants, the genomic DNA of the G418-resistant cell line was extracted, then PCR was carried out using the extracted genomic DNA, as templates, and the primers indicated below, and whether or not the EGFP and the IR/MAR sequence have been inserted into the mouse artificial chromosome (MAC) vector in a site-directed manner was confirmed at an junction region that appeared by recombination in the loxP region The primer sequences are as follows.
PCR was carried out using the ProFlex PCR System (Applied Biosystems) as a thermal cycler and KOD-FX- (Toyobo Co., Ltd.) as Taq polymerase. Buffers and dNTPs (dATP, dCTP, dGTP, and dTTP) included in the kit were used under the recommended conditions. Temperature and cycle conditions were as follows; i.e., after thermal denaturation at 95° C. for 10 minutes, a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds was conducted for 35 cycles. The 12 isolated CHO DG44 tet-O HAC clones were analyzed and found to be positive. That is, the 12 clones were evaluated to comprise the EGFP and the IR/MAR sequence inserted into tet-O HAC, and were then subjected to the subsequent analysis (
[B] Amplification of the EGFP Gene by the IR/MAR Sequence on the tet-O HAC Vector
In order to confirm that the EGFP gene was amplificed due to the integration of the IR/MAR sequence into MAC, the genome was extracted from the CHO DG44 tet-O HAC clone, and comparative analysis was performed by real-time PCR using, as a control, the CHO DG44 N10 EGFP-HAC clone with integrated single copy of the EGFP gene. (Note: the PCR analysis was not intended to measure the absolute copy number of EGFP but was intended to conduct the experiments to simply determine whether or not the EGFP was amplified in comparison with the control.)
PCR was carried out using the StepOne real-time PCR System (Applied Biosystems) as a thermal cycler and Power SYBR Green PCR master Mix (ThermoFisher) as Taq polymerase and a buffer, under the recommended conditions.
The primer sequences used are indicated below. The EGFP primers and the NV1 primers as the genomic internal standards were prepared, respectively (Nissom PM et al., Biologicals, 35, 211-5, 2007).
The 16 CHO DG44 tet-O HAC clones obtained were analyzed by real-time PCR. As a result, all the clones were found to comprise the EGFP sequences more than the control CHO DG44 N10 EGFP-HAC. Among them, 2 clones comprising particularly many EGFP sequences (i.e., cl.6-1 and cl.9-7) were subjected to the subsequent analysis (
For 2 clones selected from the above-mentioned results, two-color FISH analysis was conducted according to Matsubara et al. (FISH experimental protocols, Shujunsha Co., Ltd., Japan, 1994). The FISH analysis was carried out using mouse cot-1 DNA and pCX-EGFP plasmid as probes. As a result, both the clones were found to have larger chromosomes and many EGFP signals on human cot-1 signals, when compared with the control (CHO DG44 N10 EGFP-HAC) (
In order to confirm whether or not the EGFP expression level of the CHO DG44 tet-O HAC clones (i.e., cl.6-1 and cl.9-7) was increased accompanied with gene sequence amplification, the protein expression levels were analyzed by Western blotting.
The Western blotting was carried out using, as a cell lysis solution, RIPA buffer (Nacalai Tesque) containing a proteolytic inhibitor, and protein electrophoresis was carried out on 10% SDS acrylamide gel (Nacalai Tesque) at 2 μg clones/well using a Lapidus Mini Slab electrophoresis tank (ATTO). Proteins were transferred to a PVDF membrane with a pore size of 0.45 μm (GE Healthcare) using the XCell SureLock Mini-Cell electrophoresis system. Subsequently, a blocking reaction was carried out using a Western blocking reagent (Roche) in accordance with the recommended protocol at 4° C. overnight, the reaction product was washed 4 times with PBS-T for 10 minutes, and the resultant was subjected to the subsequent antibody reaction. EGFP detection was carried out by conducting a reaction using, as Antibody 1, the 5,000-fold diluted (0.2 ng/μl) anti-EGFP mouse antibody (abcam: ab184601) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using, as Antibody 2, the 5,000-fold diluted (0.2 ng/μl) anti-mouse IgG goat antibody (abcam: ab6789) for 45 minutes. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan). Tubulin for the internal standard was detected by conducting a reaction using the 5,000-fold diluted (0.2 ng/μl) anti-α tubulin rabbit HRP-DirecT antibody (MBL: PM054-7) for 1 hour at room temperature. After the reaction product was washed 3 times with PBS-T for 10 minutes, the subsequent reaction was carried out using the Pierce ECL Western blotting substrate (ThermoFisher) luminescence reagent in accordance with the recommended protocol. Protein detection was carried out using ImageQuant LAS4000 (FUJIFILM Corporation, Japan).
As a result of Western blot analysis, higher protein expression levels were observed in the CHO DG44 tet-O HAC clones, when compared with the control (
According to the present invention, a mammalian cell transformed with the IR/MAR-MMAC nucleic acid construct comprising DNA encoding a gene of interest is cultured, thereby enabling stable and high expression of the gene of interest.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/JP2018/040852 | 11/2/2018 | WO | 00 |