WO2022196245A1 - Selection marker expression cassette - Google Patents

Selection marker expression cassette Download PDF

Info

Publication number
WO2022196245A1
WO2022196245A1 PCT/JP2022/006667 JP2022006667W WO2022196245A1 WO 2022196245 A1 WO2022196245 A1 WO 2022196245A1 JP 2022006667 W JP2022006667 W JP 2022006667W WO 2022196245 A1 WO2022196245 A1 WO 2022196245A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
expression
sequence dna
dna
selectable marker
Prior art date
Application number
PCT/JP2022/006667
Other languages
French (fr)
Japanese (ja)
Inventor
▲隆▼之 松岡
武志 林
Original Assignee
株式会社chromocenter
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社chromocenter filed Critical 株式会社chromocenter
Priority to JP2023506894A priority Critical patent/JP7416355B2/en
Publication of WO2022196245A1 publication Critical patent/WO2022196245A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a selectable marker expression cassette, in particular, a selectable marker expression cassette that enables efficient selection of cultured animal cells that highly express a target protein.
  • Methods for attenuating the expression or function of the selectable marker include using a promoter with weak transcriptional activity, introducing an mRNA destabilizing sequence into the expression cassette of the selectable marker, and introducing mutations into the coding sequence of the selectable marker. and a method of attenuating the function of the selectable marker protein itself.
  • upstream ORF upstream open reading frame
  • uORF upstream open reading frame
  • the upstream ORF exists in the 5' untranslated region of mRNA and regulates the translation of the downstream ORF by various mechanisms.It is known that the translation of the upstream ORF generally reduces the translation efficiency of the downstream ORF.
  • Pattern 1 is classified into the following subclasses. Pattern 1-1: After termination of translation of the upstream ORF, the ribosome leaves the mRNA and the downstream ORF is not translated. Pattern 1-2: After translation termination of the upstream ORF, the ribosome recognizes the initiation codon of the downstream ORF and initiates translation without leaving the mRNA. Patterns 1-4: After termination of translation of the upstream ORF, the ribosome does not leave the mRNA, but passes through the initiation codon of the downstream ORF, so the downstream ORF is not translated.
  • Pattern 2 is classified into the following subclasses. Pattern 2-1: Recognizes the initiation codon of the downstream ORF and initiates translation. Pattern 2-2: Since the initiation codon of the downstream ORF is also passed through, the downstream ORF is not translated.
  • the present inventors studied a technique for (1) increasing the recognition efficiency of initiation codons of upstream ORFs and (2) decreasing the recognition efficiency of initiation codons of downstream ORFs.
  • the modified Kozak sequence DNA and the short ORF (upstream ORF) sequence DNA upstream of the gene whose expression is to be attenuated we succeeded in effectively attenuating the expression of the selectable marker gene.
  • the present invention has been completed.
  • An object of the present invention is to comprising an expression control sequence DNA,
  • the expression control sequence DNA comprises one or more secondary expression control sequence DNAs,
  • the secondary expression regulatory sequence DNA comprises a modified Kozak sequence DNA and an upstream ORF sequence DNA downstream of the modified Kozak sequence DNA,
  • the base sequence of the secondary expression regulatory sequence DNA has only one start codon base sequence, To provide a selectable marker expression cassette.
  • selectable marker expression cassette By using the selectable marker expression cassette according to the present invention, it is possible to attenuate the expression of the selectable marker and reduce the translation efficiency of the selectable marker gene.
  • the one or more secondary expression regulatory sequence DNAs may be one, two or three secondary expression regulatory sequence DNAs.
  • the upstream ORF sequence DNA has a nucleotide sequence represented by SEQ ID NO: 1 or 2, or its base sequence, 1) A nucleotide sequence with 95% or more sequence homology, or 2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or 3) It may consist of a nucleotide sequence capable of hybridizing with a complementary sequence under stringent conditions.
  • the modified Kozak sequence DNA has the base sequence represented by SEQ ID NO: 3, or its base sequence; 1) A nucleotide sequence with 95% or more sequence homology, or 2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or 3) It may consist of a nucleotide sequence capable of hybridizing with a complementary sequence under stringent conditions.
  • Another object of the invention is to comprising the selectable marker expression cassette; It is to provide an expression vector.
  • the expression vector of the present invention it is possible to attenuate the expression of the selectable marker and reduce the translation efficiency of the selectable marker gene.
  • the expression vector may further comprise two terminal tDNA insulators.
  • the selectable marker expression cassette may be located between the two terminal tDNA insulators.
  • the expression vector may further comprise one or more target protein expression cassettes.
  • Said one or more protein-of-interest expression cassettes may be located between said two terminal tDNA insulators.
  • the expression vector may further comprise one or more intermediate tDNA insulators.
  • the one or more protein-of-interest expression cassettes may be a plurality of protein-of-interest expression cassettes. At least one of said intermediate tDNA insulators may be present between one of said plurality of protein of interest expression cassettes and an adjacent protein of interest expression cassette.
  • the intermediate tDNA insulator may have a nucleotide sequence derived from the mouse tRNA gene.
  • the terminal tDNA insulator may have a base sequence derived from the mouse tRNA gene.
  • the selectable marker expression cassette may further comprise a selectable marker gene.
  • the selectable marker gene may be located downstream of the expression control sequence DNA.
  • the start codon sequence of the selectable marker gene may be included in the base sequence of the upstream ORF sequence DNA.
  • FIG. 1 shows a schematic diagram of the expression vector preparation plasmid (pCHR002) used in this example.
  • FIG. 2 shows a schematic diagram of the second gene cloning plasmid (pCHR006) used in this example.
  • FIG. 3 shows a schematic diagram of the vector plasmid (pCHR002-Lc) in which the Herceptin light chain gene was introduced into the expression vector preparation plasmid (pCHR002).
  • FIG. 4 shows a schematic diagram of the vector plasmid (CHR008) in which the Herceptin heavy chain gene was introduced into the second gene cloning plasmid (pCHR006).
  • FIG. 1 shows a schematic diagram of the expression vector preparation plasmid (pCHR002) used in this example.
  • FIG. 2 shows a schematic diagram of the second gene cloning plasmid (pCHR006) used in this example.
  • FIG. 3 shows a schematic diagram of the vector plasmid (pCHR002-Lc)
  • FIG. 5 shows a schematic diagram of a Herceptin expression vector (pCHR012) in which a fragment containing the Herceptin heavy chain gene derived from the vector plasmid (pCHR008) shown in FIG. 4 was introduced into the vector plasmid (pCHR002-Lc) shown in FIG. ing.
  • FIG. 6 shows a schematic diagram of the positions of the modified Kozak sequence and the upstream ORF in the vector plasmid used in this example, and the alignment arrangement of the base sequences of the modified Kozak sequence (underlined) and the upstream ORF (box). .
  • FIG. 7 shows a schematic diagram of the Herceptin expression vector (pELC2+HC) in which the Herceptin gene was introduced into the Mammalian PowerExpress System (registered trademark) (TOYOBO).
  • FIG. 8 shows a graph of the amount of herceptin secreted from each cell transfected with pCHR012, pCHR042, pCHR067, pCHR068, pCHR069 and pELC2+HC.
  • FIG. 9 shows a graph of the amount of Herceptin secreted from each cell transfected with pELC2+HC, pCHR042, and pCHR069.
  • nucleotide sequences in this specification, drawings and sequence listing are listed in order from the 5' end to the 3' end.
  • 100th base means the 100th base from the 5' end.
  • reverse complementary sequence means a sequence that is in a reverse complementary relationship to a given sequence.
  • the reverse complement of the 5'-AAATTCGG-3' sequence is 5'-CCGAATTT-3'.
  • upstream refers to the direction of the 5' end
  • downstream means the direction of the 3' end.
  • the initiation codon refers to a codon that specifies the initiation of protein synthesis, and is usually represented by the nucleotide sequence ATG or AUG.
  • the nucleotide sequence of the nucleic acid molecule (e.g., DNA, region, cassette, genome, and vector plasmid) according to this embodiment includes the nucleotide sequence, 1) is a nucleotide sequence with 95% or more sequence homology, or 2) a nucleotide sequence in which one or several bases have been deleted, substituted, or added, or 3) It may be a nucleotide sequence that can hybridize with a complementary sequence under stringent conditions.
  • a nucleotide sequence having sequence homology with the nucleotide sequence shown in the SEQ ID NO may have a sequence homology of 95% or more, for example, 96% or more, 97% or more, 98% or more, 99% or more, It may be 99.5% or more, or 99.8% or more.
  • “several bases” in “a base sequence in which one or several bases are deleted, substituted, or added” are, for example, 2 to 10 bases, 2 to 9 bases, 2 to 8 bases, 2 to 7 base, 2-6 bases, 2-5 bases, 2-4 bases, 2-3 bases, and 2 bases.
  • the number of deleted, substituted or added bases is generally preferably as small as possible. Two or more of deletion, substitution, and addition of bases may occur simultaneously.
  • well-known techniques can be used for deletion, substitution, and addition of bases.
  • stringent conditions include, for example, the following (1) and (2).
  • Low ionic strength and high washing temperature include, for example, 0.015 M NaCl/0.0015 M sodium citrate (titrate)/0.1% SDS at a temperature of 50°C. or (2) using a denaturing agent such as formamide during hybridization, e.g., 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyinylpyrrolidone/ 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at a temperature of 42°C.
  • a denaturing agent such as formamide during hybridization, e.g., 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyinylpyrrolidone/ 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at a temperature of 42°C.
  • Another example is 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, Sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, 10% dextran sulfate, temperature 42° C., washing temperature 42° C., 0.2 ⁇ SSC, 0.1% SDS. It will also be appreciated by those of ordinary skill in the art that stringent conditions can be altered as appropriate to obtain a clear and detectable hybridization signal.
  • molecular biology techniques e.g., cloning, plasmid extraction, DNA fragment cleavage, ligation, hybridization, site-directed mutagenesis, PCR, Western blotting, etc.
  • Conventional methods well known to those skilled in the art can be employed. For these methods, see Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, (1989), etc. .
  • the selectable marker expression cassette according to this embodiment is comprising an expression control sequence DNA,
  • the expression control sequence DNA comprises one or more secondary expression control sequence DNAs,
  • the secondary expression regulatory sequence DNA comprises a modified Kozak sequence DNA and an upstream ORF sequence DNA downstream of the modified Kozak sequence DNA,
  • the base sequence of the secondary expression regulatory sequence DNA has only one start codon base sequence.
  • the base sequence of Kozak sequence DNA is a common sequence that appears in eukaryotic mRNA and is mainly involved in the initiation of translation. However, it is not a strict consensus sequence and very often there are discrepancies.
  • the Kozak sequence of vertebrates is represented by (gcc)gccRccAUGG (SEQ ID NO: 39). considered to play an important role (Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987;15(20):8125-8148. doi:10.1093/nar/15.20 .8125).
  • Kozak sequences (1) lower case letters indicate the most common base at that position, although bases may vary, (2) "AUGG" sequences indicate highly conserved bases, and (3) Sequences in brackets (gcc) are of uncertain significance.
  • the modified Kozak sequence DNA in this embodiment has a base sequence different from that of the Kozak sequence DNA.
  • the above-mentioned modified Kozak sequence DNA has a base sequence represented by SEQ ID NO: 3 (GCCACC), or a mutant base sequence thereof (the base sequence and 1) A nucleotide sequence with 95% or more sequence homology, or 2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or 3) A nucleotide sequence that can hybridize with a complementary sequence under stringent conditions) It may consist of The nucleotide sequence of the modified Kozak sequence DNA does not contain an ATG sequence that serves as an initiation codon.
  • the upstream ORF sequence DNA in this embodiment is located downstream of the modified Kozak sequence DNA.
  • the upstream ORF sequence DNA has only one initiation codon (ATG).
  • the secondary expression control sequence DNA has only one initiation codon.
  • the initiation codon (ATG) of the upstream ORF sequence DNA is located at its 5' end.
  • the nucleotide sequence of the upstream ORF sequence DNA may have an ATG sequence downstream of the initiation codon (ATG) that does not serve as an initiation codon for the upstream ORF.
  • the upstream ORF sequence DNA has a base sequence represented by SEQ ID NO: 1 (ATGGCTTGA) or SEQ ID NO: 2 (ATGGCTCTAGAAATCGGATGA), or a variant base sequence thereof (the base sequence and 1) A nucleotide sequence with 95% or more sequence homology, or 2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or 3) A nucleotide sequence that can hybridize with a complementary sequence under stringent conditions) It may consist of The base sequence of the upstream ORF sequence DNA does not include the base sequence represented by SEQ ID NO: 3 or its mutant base sequence.
  • the upstream ORF sequence DNA is directly linked to the modified Kozak sequence DNA (in other words, no bases are present between the upstream ORF sequence DNA and the modified Kozak sequence DNA).
  • the upstream ORF sequence DNA is linked to the modified Kozak sequence DNA via spacer DNA.
  • the spacer DNA may consist of 1, 2, 3, 4 or 5 arbitrary bases. Spacer DNA does not have an ATG sequence that serves as an initiation codon.
  • the selectable marker expression cassette may comprise a promoter (eg mouse PGK promoter) and/or a poly A signal (eg BGH poly A signal) in addition to the expression control sequence DNA.
  • the selection marker expression cassette includes a fluorescent selection marker gene (e.g., DNA encoding green fluorescent protein (GFP)), a drug selection marker gene (e.g., puromycin resistance gene and neomycin resistance gene), and/or a nutritional selection marker.
  • GFP green fluorescent protein
  • Selectable marker genes including genes eg, glutamine synthetase gene
  • the selectable marker expression cassette may comprise a promoter, expression control sequence DNA, selectable marker gene and poly A signal.
  • the expression level of the "selective marker gene” is 0.5%, 1%, 5% compared to the expression level of the "selective marker gene” that does not employ the configuration of this embodiment (not subjected to expression suppression). %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more.
  • the expression control sequence DNA comprises one or more secondary expression control sequence DNAs.
  • one secondary expression control sequence DNA is directly connected to adjacent secondary expression control sequence DNAs.
  • one secondary expression control sequence DNA is directly connected to adjacent secondary expression control sequence DNAs via spacer DNAs.
  • the spacer DNA may consist of 1, 2, 3, 4 or 5 arbitrary bases. Spacer DNA does not have an ATG sequence that serves as an initiation codon.
  • the one or more secondary expression control sequence DNAs are 1, 2 or 3 secondary expression control sequence DNAs.
  • the expression control sequence DNA may comprise one secondary expression control sequence DNA.
  • the initiation codon sequence of the selectable marker gene may be included (present) in the nucleotide sequence of the upstream ORF sequence DNA.
  • the reading frame of the upstream ORF sequence DNA may overlap the start codon of the selectable marker gene.
  • the Kozak sequence, modified Kozak sequence and their mutant base sequences are not present between the selectable marker gene and the adjacent upstream ORF sequence.
  • the expression vector according to this embodiment comprises a selectable marker expression cassette.
  • the expression vector in this embodiment can be created by inserting a selection marker expression cassette into a cloning vector.
  • cloning vectors include plasmids, phages, phagemids, cosmids, fosmids, artificial chromosomes, and the like. Specifically, E.
  • coli-derived plasmids e.g., pBR322, pBR325, pUC12, pUC13
  • yeast-derived plasmids e.g., pSH19, pSH15
  • Bacillus subtilis-derived plasmids e.g., pUB110, pTP5, pC194
  • animal cell expression plasmids e.g., pA1 -11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo
  • bacteriophages such as ⁇ phage
  • viral vectors such as HIV, adenovirus, retrovirus, vaccinia virus, baculovirus, and artificial chromosomes be able to.
  • the expression vector according to this embodiment may further comprise two terminal tDNA insulators.
  • the selection marker expression cassette is preferably positioned between the two terminal tDNA insulators.
  • a terminal tDNA insulator is a DNA consisting of nucleotide sequences capable of delimiting expression units of a gene, which reduces promoter interference, silencing, and is involved in stabilizing gene expression, for example, consisting of the nucleotide sequence of the mouse tRNA gene. DNA can be mentioned.
  • the expression vector according to this embodiment can comprise one or more target protein expression cassettes.
  • the expression vector according to this embodiment comprises one or more protein-of-interest expression cassettes
  • the one or more protein-of-interest expression cassettes are preferably located between the two terminal tDNA insulators.
  • a protein of interest expression cassette comprises a gene that expresses the protein of interest.
  • the target protein expression cassette includes, in addition to the gene expressing the target protein, (For example, it may have at least one selected from the group consisting of a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE) sequence and a poly A signal (SV40 poly A signal).
  • WPRE Woodchuck hepatitis virus Post-transcriptional Regulatory Element
  • Target protein refers to a protein whose expression level is to be increased by attenuating the expression level of the "selection marker gene”.
  • the expression level of the "target protein” in the configuration of this embodiment is compared with the expression level of the "target protein” that does not employ the configuration of the present invention (the selection marker is not suppressed in expression). 5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold or more than 5.0-fold.
  • the expression vector according to this embodiment can comprise one or more intermediate tDNA insulators.
  • the expression vector according to this embodiment comprises a plurality of target protein expression cassettes
  • at least one intermediate tDNA insulator is present between one of the plurality of target protein expression cassettes and the adjacent target protein expression cassette.
  • the intermediate tDNA insulator may be the same as the intermediate tDNA insulator, and may include, for example, DNA consisting of the base sequence of the mouse tRNA gene.
  • Example 1 Construction of expression vector construction plasmid (pCHR002) and second gene cloning plasmid (pCHR006) Plasmid constructed by VectorBuilder, consisting of standard components available on VectorBuilder's online platform (Vector ID: VB900085-9593ncv and VB190621-1088bqm) and a plasmid containing artificially synthesized tDNA (Ebersole T, Kim JH, Samoshkin A, et al. tRNA genes protect a reporter gene from epigenetic silencing in mouse cells. Cell Cycle. 2011;10(16):2779 -2791.) was prepared. Based on these plasmids, the components shown in Table 1 were amplified by PCR so that the restriction enzyme sites shown in FIG. 1 were added.
  • Each component was amplified by PCR using the primers shown in Table 2.
  • pCHR002 contains tDNA, human EF-1 ⁇ promoter, SV40 late pA, mPGK promoter, PuroR, BGH pA, bacterial origin of replication (pUC ori), ampicillin resistance marker (AmpR).
  • a second gene cloning plasmid pCHR006 containing tDNA, human EF-1 ⁇ promoter, SV40 late pA, pUC ori, and AmpR was constructed (Fig. 2). Each component was amplified by PCR using the primers shown in Table 3.
  • the bound DNA fragment was amplified by PCR using the primer set AscI_MluI_NdeI_tDNA-F (SEQ ID NO: 20) and NotI_PacI_EcoT22I_SV40pA-R (SEQ ID NO: 21).
  • the amplified ligated DNA fragment was introduced into the restriction enzyme AscI and NotI sites of the backbone of a plasmid (VB900085-9593ncv) constructed by VectorBuilder to construct pCHR006.
  • Herceptin Expression Vector Herceptin light chain gene (SEQ ID NO: 33) and Herceptin heavy chain gene (SEQ ID NO: :34) was amplified by PCR, and the primers used are shown in Table 4.
  • the Herceptin light chain gene was amplified by PCR to add HindIII and SalI restriction enzyme sites.
  • pCHR002-Lc was constructed by introducing the amplified Herceptin light chain gene into the HindIII and SalI sites of pCHR002 (Fig. 3).
  • the Herceptin heavy chain gene was amplified by PCR to add HindIII and SbfI restriction enzyme sites.
  • pCHR008 was constructed by introducing the amplified Herceptin heavy chain gene into the HindIII and SbfI sites of pCHR006 (Fig. 4).
  • a fragment containing the Herceptin heavy chain gene was excised from pCHR008 with restriction enzymes MluI and NsiI.
  • Herceptin expression vector pCHR012 was constructed by introducing the excised fragment into the MluI and NsiI sites of pCHR002-Lc (Fig. 5).
  • pCHR027 Using pCHR027 as a template, a PCR product amplified with a primer set of PacI_mPGKpro-F (SEQ ID NO: 10) and XbaI-K-uORF-mPGK-R (SEQ ID NO: 35) was introduced into the PacI and XbaI sites of pCHR027. This resulted in the construction of pCHR042 with an upstream ORF inserted into the puromycin resistance gene (Fig. 6). Similarly, the plasmids shown in Table 5 were constructed by introducing the PCR products amplified using the corresponding primer sets into the PacI and XbaI sites of pCHR027.
  • pCHR067 has two upstream ORFs inserted into the puromycin resistance gene
  • pCHR068 has three upstream ORFs inserted into the puromycin resistance gene
  • pCHR069 has an insertion over the start codon of the puromycin resistance gene.
  • Upstream ORF is inserted to wrap.
  • CHO-G1 Chinese hamster ovary-derived cells CHO-G1 (established by chromocenter, Inc.) adapted to suspension culture were placed in a 12-well plate. 1 mL each was seeded. CHO-G1 cells were prepared for transfection by overnight adherent culture.
  • ProCHO4 medium with 5% FBS HT Supplement (1 ⁇ ) (GIBCO), L-Glutamine (2 mM) (GIBCO), ProCHO4 Protein supplemented with Penicillin-Streptomycin (100 U/mL) (GIBCO) -free CHO Medium (manufactured by LONZA) was used as the medium.
  • plasmids As plasmids, pCHR012, pCHR042, pCHR067, pCHR068, pCHR069, pELC2+HC (Fig. 7, Herceptin expression vector in which artificially synthesized Herceptin gene was introduced into TOYOBO, Mammalian PowerExpress System (registered trademark)) was used. Transfection was performed by the following method. 1.6 ⁇ g of plasmid vector was diluted with 100 ⁇ L Opti-MEM I Reduced Serum Medium (manufactured by GIBCO).
  • the dispersed cells were transferred to a ⁇ 93 ⁇ 19.2 mm cell culture dish and cultured in ProCHO4 medium+5% FBS. The next day, Puromycin (manufactured by Sigma-Aldrich) was added to the medium in which the cells were being cultured at a concentration of 7.5 ⁇ g/mL, and drug-selective adhesion culture was performed for 6 to 12 days. During the selection culture, the medium was changed every 3-4 days, or subculture was performed at an appropriate cell density. When the cells had grown sufficiently, they were transferred to stationary stationary culture in ProCHO4 medium containing 5 ⁇ g/mL Puromycin. The cells were further cultured in floating stationary culture for about one week to obtain a CHO-G1 heterocell pool stably expressing Herceptin.
  • Puromycin manufactured by Sigma-Aldrich
  • Herceptin Productivity Each herceptin stably expressing CHO-G1 heterocell pool transfected with pCHR012, pCHR042, pCHR067, ppCH068, CHR069, and pELC2+HC expression vectors was cultured in fresh ProCHO4 medium containing 5 ⁇ g/mL Puromycin. It was adjusted to ⁇ 10 5 cells/mL. 2 mL of the cell pool was seeded in a 6-well plate and cultured in suspension at 37°C. After 5 days, the culture supernatant was collected, centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected to obtain a sample of produced Herceptin. Herceptin was quantified with an Ensight Multimode Plate Reader (PerkinElmer) using AlphaLISA human IgG kit (PerkinElmer).
  • Fig. 8 shows the results of herceptin quantification in the culture supernatant of each herceptin-stably-expressing CHO-G1 heterocell pool into which each expression vector of pCHR012, pCHR042, pCHR067, pCHR068, pCHR069, and pELC2+HC was introduced.
  • the vector (pCHR042) in which the upstream ORF was inserted into the puromycin resistance gene increased herceptin production by about 3-fold compared to the vector (pCHR012) in which the upstream ORF was not inserted.
  • the vector (pCHR067) loaded with two copies of the upstream ORF further increased herceptin production compared to the vector (pCHR042).
  • the vector (pCHR069) in which the upstream ORF was inserted so as to overlap with the initiation codon of the puromycin resistance gene produced Herceptin at the same level as the vector (pCHR067).
  • CHO-K1 cells (JCRB cell bank, IFO50414) were adapted to suspension culture at chromocenter.
  • CHO-K1 cells for transfection were prepared and transfected with pELC2+HC, pCHR042, and pCHR069 using the same method as for the CHO-G1 cells described above.
  • drug-selective adhesion culture was performed for 7 days, and when the cells were sufficiently grown, they were transferred to suspension stationary culture in ProCHO4 medium containing 5 ⁇ g/mL Puromycin. Cell culture was further carried out for about 2 weeks in stationary stationary culture to obtain a CHO-K1 heterocell pool stably expressing Herceptin.
  • FIG. 9 shows the results of quantification of herceptin in the culture supernatant of each CHO-K1 heterocell pool stably expressing herceptin into which each expression vector of pELC2+HC, pCHR042 and pCHR069 was introduced.
  • pCHR042 showed about 2.8-fold higher herceptin production and pCHR069 about 4.5-fold higher than pELC2+HC.

Abstract

There has been a problem that the translation efficiency of a downstream ORF cannot be reduced to a practical level even when an upstream ORF is present. The purpose of the present invention is to provide a selection marker expression cassette comprising expression regulation sequence DNA, in which the expression regulation sequence DNA includes at least one expression regulation sub-sequence DNA, the expression regulation sub-sequence DNA includes modified Kozak sequence DNA and upstream ORF sequence DNA located downstream of the modified Kozak sequence DNA, and the nucleotide sequence for the expression regulation sub-sequence DNA includes only one nucleotide sequence for an initiation codon.

Description

選択マーカー発現カセットSelectable marker expression cassette
　本発明は、選択マーカー発現カセット、特に、目的タンパク質を高発現する動物培養細胞を効率的に選抜することが可能な選択マーカー発現カセットに関する。 The present invention relates to a selectable marker expression cassette, in particular, a selectable marker expression cassette that enables efficient selection of cultured animal cells that highly express a target protein.
　これまで目的タンパク質を高レベルで発現する細胞を効率的に選抜することを目的に、選択マーカーの発現や機能を減弱化することに基づく様々な方法が開発されてきた。選択マーカーの発現や機能を減弱化する方法としては、転写活性の弱いプロモーターを利用する方法、選択マーカーの発現カセットにmRNA不安定化配列を導入する方法、選択マーカーのコード配列に変異を導入して選択マーカータンパク質自体の機能を減弱化する方法などが挙げられる。 To date, various methods have been developed based on attenuating the expression and function of selectable markers with the aim of efficiently selecting cells that express a high level of a target protein. Methods for attenuating the expression or function of the selectable marker include using a promoter with weak transcriptional activity, introducing an mRNA destabilizing sequence into the expression cassette of the selectable marker, and introducing mutations into the coding sequence of the selectable marker. and a method of attenuating the function of the selectable marker protein itself.
　不必要なタンパク質の発現や機能を減弱化する方法の一つに、Upstream open reading frame（上流ORF又はuORF)を利用した方法が存在する。上流ORFは、mRNAの5'非翻訳領域に存在し、下流のORFの翻訳を様々な機構で制御するが、一般に上流ORFの翻訳は下流のORFの翻訳効率を低下させることが知られている(非特許文献１及び２)。 One of the methods for attenuating the expression and function of unnecessary proteins is to use the upstream open reading frame (upstream ORF or uORF). The upstream ORF exists in the 5' untranslated region of mRNA and regulates the translation of the downstream ORF by various mechanisms.It is known that the translation of the upstream ORF generally reduces the translation efficiency of the downstream ORF. (Non-Patent Documents 1 and 2).
　しかしながら、上流ORFが存在する場合、上流ORFの開始コドンがリボソームによって認識されて翻訳される場合（パターン１）とリボソームが上流ORFを通過する場合（パターン２）が存在していた。 However, when there was an upstream ORF, there were cases where the initiation codon of the upstream ORF was recognized and translated by the ribosome (pattern 1) and cases where the ribosome passed through the upstream ORF (pattern 2).
　パターン１は、以下のサブクラスに分類される。
パターン１-１：上流ORFの翻訳終結後、リボソームがmRNAから離れ、下流ORFが翻訳されない。
パターン１-２：上流ORFの翻訳終結後、リボソームがmRNAから離れることなく下流ORFの開始コドンを認識して翻訳が開始される。
パターン１-４：上流ORFの翻訳終結後、リボソームがmRNAから離れないが、下流ORFの開始コドンを通過するため、下流ORFは翻訳されない。 Pattern 1 is classified into the following subclasses.
Pattern 1-1: After termination of translation of the upstream ORF, the ribosome leaves the mRNA and the downstream ORF is not translated.
Pattern 1-2: After translation termination of the upstream ORF, the ribosome recognizes the initiation codon of the downstream ORF and initiates translation without leaving the mRNA.
Patterns 1-4: After termination of translation of the upstream ORF, the ribosome does not leave the mRNA, but passes through the initiation codon of the downstream ORF, so the downstream ORF is not translated.
　パターン２は、以下のサブクラスに分類される。
パターン２-１：下流ORFの開始コドンを認識して翻訳を開始する。
パターン２-２：下流ORFの開始コドンも通過するため、下流ORFは翻訳されない。 Pattern 2 is classified into the following subclasses.
Pattern 2-1: Recognizes the initiation codon of the downstream ORF and initiates translation.
Pattern 2-2: Since the initiation codon of the downstream ORF is also passed through, the downstream ORF is not translated.
　このように、上流ORFが存在していても、下流のORFの翻訳効率を実用的な水準で低下させることができない問題が存在していた。即ち、この技術では、目的タンパク質を高レベルで発現することを目的に、選択マーカーの発現や機能を効果的に減弱化することができなかった。 Thus, even if there is an upstream ORF, there is a problem that the translation efficiency of the downstream ORF cannot be reduced to a practical level. That is, this technique cannot effectively attenuate the expression or function of the selectable marker for the purpose of expressing the target protein at a high level.
　本発明者らは、下流ORFの翻訳効率を低下させるために、（１）上流ORFの開始コドンの認識効率を高め且つ（２）下流ORFの開始コドンの認識効率を低下させる技術を研究した。その結果、改変型Kozak配列DNA及び短いORF（上流ORF）配列DNAを、発現を減弱化させたい遺伝子の上流に挿入することによって、選択マーカー遺伝子の発現を効果的に減弱化することに成功し、本発明は完成した。 In order to reduce the translation efficiency of downstream ORFs, the present inventors studied a technique for (1) increasing the recognition efficiency of initiation codons of upstream ORFs and (2) decreasing the recognition efficiency of initiation codons of downstream ORFs. As a result, by inserting the modified Kozak sequence DNA and the short ORF (upstream ORF) sequence DNA upstream of the gene whose expression is to be attenuated, we succeeded in effectively attenuating the expression of the selectable marker gene. , the present invention has been completed.
　本発明の目的は、
　発現調節配列DNAを備え、
　上記発現調節配列DNAは、１又は複数の副発現調節配列DNAを備え、
　上記副発現調節配列DNAは、改変型Kozak配列DNAと上記改変型Kozak配列DNAの下流に上流ORF配列DNAとを備え、
　上記副発現調節配列DNAの塩基配列は、開始コドンの塩基配列を１つだけ備える、
　選択マーカー発現カセット
を提供することである。 An object of the present invention is to
comprising an expression control sequence DNA,
The expression control sequence DNA comprises one or more secondary expression control sequence DNAs,
The secondary expression regulatory sequence DNA comprises a modified Kozak sequence DNA and an upstream ORF sequence DNA downstream of the modified Kozak sequence DNA,
The base sequence of the secondary expression regulatory sequence DNA has only one start codon base sequence,
To provide a selectable marker expression cassette.
　本発明にかかる選択マーカー発現カセットを用いることで、選択マーカーの発現を減弱化させ、選択マーカー遺伝子の翻訳効率を低下させることができる。 By using the selectable marker expression cassette according to the present invention, it is possible to attenuate the expression of the selectable marker and reduce the translation efficiency of the selectable marker gene.
　上記１又は複数の副発現調節配列DNAは、１つ、２つ又は３つの副発現調節配列DNAであってもよい。 The one or more secondary expression regulatory sequence DNAs may be one, two or three secondary expression regulatory sequence DNAs.
　上記上流ORF配列DNAは、配列番号：１又は２で表される塩基配列、
又は
その塩基配列と、
1）９５％以上の配列相同性を有する塩基配列、又は、
2）１または数塩基が欠失、置換、付加されている塩基配列、又は、
3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列
からなるものであってもよい。 The upstream ORF sequence DNA has a nucleotide sequence represented by SEQ ID NO: 1 or 2,
or its base sequence,
1) A nucleotide sequence with 95% or more sequence homology, or
2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
3) It may consist of a nucleotide sequence capable of hybridizing with a complementary sequence under stringent conditions.
　上記改変型Kozak配列DNAは、配列番号：３で表される塩基配列、
又は、
その塩基配列と、
1）９５％以上の配列相同性を有する塩基配列、又は、
2）１または数塩基が欠失、置換、付加されている塩基配列、又は、
3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列
からなるものであってもよい。 The modified Kozak sequence DNA has the base sequence represented by SEQ ID NO: 3,
or
its base sequence;
1) A nucleotide sequence with 95% or more sequence homology, or
2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
3) It may consist of a nucleotide sequence capable of hybridizing with a complementary sequence under stringent conditions.
　本発明の別の目的は、
上記選択マーカー発現カセットを備える、
　発現ベクターを提供することである。 Another object of the invention is to
comprising the selectable marker expression cassette;
It is to provide an expression vector.
　本発明にかかる発現ベクターを用いることで、選択マーカーの発現を減弱化させ、選択マーカー遺伝子の翻訳効率を低下させることができる。 By using the expression vector of the present invention, it is possible to attenuate the expression of the selectable marker and reduce the translation efficiency of the selectable marker gene.
　上記発現ベクターは、２つの末端tDNAインシュレーターを更に備えていてもよい。上記選択マーカー発現カセットは、上記２つの末端tDNAインシュレーターの間に位置してもよい。 The expression vector may further comprise two terminal tDNA insulators. The selectable marker expression cassette may be located between the two terminal tDNA insulators.
　上記発現ベクターは、１又は複数の目的タンパク質発現カセットを更に備えていてもよい。上記１又は複数の目的タンパク質発現カセットは、上記２つの末端tDNAインシュレーターの間に位置してもよい。 The expression vector may further comprise one or more target protein expression cassettes. Said one or more protein-of-interest expression cassettes may be located between said two terminal tDNA insulators.
　上記発現ベクターは、更に、１又は複数の中間tDNAインシュレーターを備えていてもよい。上記１又は複数の目的タンパク質発現カセットは、複数の目的タンパク質発現カセットであってもよい。少なくとも１の上記中間tDNAインシュレーターが、上記複数の目的タンパク質発現カセットのうちの１つと隣接する目的タンパク質発現カセットとの間に存在していてもよい。 The expression vector may further comprise one or more intermediate tDNA insulators. The one or more protein-of-interest expression cassettes may be a plurality of protein-of-interest expression cassettes. At least one of said intermediate tDNA insulators may be present between one of said plurality of protein of interest expression cassettes and an adjacent protein of interest expression cassette.
　上記中間tDNAインシュレーターは、マウスtRNA遺伝子由来の塩基配列を有してもよい。 The intermediate tDNA insulator may have a nucleotide sequence derived from the mouse tRNA gene.
　上記末端tDNAインシュレーターは、マウスtRNA遺伝子由来の塩基配列を有してもよい。 The terminal tDNA insulator may have a base sequence derived from the mouse tRNA gene.
　上記選択マーカー発現カセットは、選択マーカー遺伝子を更に備えていてもよい。上記選択マーカー遺伝子は、上記発現調節配列DNAの下流に位置してもよい。 The selectable marker expression cassette may further comprise a selectable marker gene. The selectable marker gene may be located downstream of the expression control sequence DNA.
　上記上流ORF配列DNAの塩基配列が配列番号：２からなる場合、上記選択マーカー遺伝子の開始コドン配列は、上記上流ORF配列DNAの塩基配列中に含まれていてもよい。 When the base sequence of the upstream ORF sequence DNA consists of SEQ ID NO: 2, the start codon sequence of the selectable marker gene may be included in the base sequence of the upstream ORF sequence DNA.
図１は、本実施例で使用する発現ベクター作製用プラスミド（pCHR002）の模式図を示している。FIG. 1 shows a schematic diagram of the expression vector preparation plasmid (pCHR002) used in this example. 図２は、本実施例で使用する第2遺伝子クローニング用プラスミド（pCHR006）の模式図を示している。FIG. 2 shows a schematic diagram of the second gene cloning plasmid (pCHR006) used in this example. 図３は、発現ベクター作製用プラスミド（pCHR002）にハーセプチン軽鎖遺伝子が導入されたベクタープラスミド（pCHR002-Lc）の模式図を示している。FIG. 3 shows a schematic diagram of the vector plasmid (pCHR002-Lc) in which the Herceptin light chain gene was introduced into the expression vector preparation plasmid (pCHR002). 図４は、第2遺伝子クローニング用プラスミド（pCHR006）にハーセプチン重鎖遺伝子が導入されたベクタープラスミド（CHR008）の模式図を示している。FIG. 4 shows a schematic diagram of the vector plasmid (CHR008) in which the Herceptin heavy chain gene was introduced into the second gene cloning plasmid (pCHR006). 図５は、図３に示すベクタープラスミド（pCHR002-Lc）に、図４に示すベクタープラスミド（pCHR008）由来のハーセプチン重鎖遺伝子を含む断片が導入されたハーセプチン発現ベクター（pCHR012）の模式図を示している。FIG. 5 shows a schematic diagram of a Herceptin expression vector (pCHR012) in which a fragment containing the Herceptin heavy chain gene derived from the vector plasmid (pCHR008) shown in FIG. 4 was introduced into the vector plasmid (pCHR002-Lc) shown in FIG. ing. 図６は、本実施例で使用するベクタープラスミドにおける改変型コザック配列及び上流ORFの位置並びに改変型コザック配列（下線）及び上流ORF（四角枠）の塩基配列を整列配置した模式図を示している。FIG. 6 shows a schematic diagram of the positions of the modified Kozak sequence and the upstream ORF in the vector plasmid used in this example, and the alignment arrangement of the base sequences of the modified Kozak sequence (underlined) and the upstream ORF (box). . 図７は、Mammalian PowerExpress System（登録商標）（TOYOBO）にハーセプチン遺伝子を導入したハーセプチン発現ベクター（pELC2+HC）の模式図を示している。FIG. 7 shows a schematic diagram of the Herceptin expression vector (pELC2+HC) in which the Herceptin gene was introduced into the Mammalian PowerExpress System (registered trademark) (TOYOBO). 図８は、pCHR012、pCHR042、pCHR067、pCHR068、pCHR069及びpELC2+HCを導入した各細胞から分泌されたハーセプチン量のグラフを示している。FIG. 8 shows a graph of the amount of herceptin secreted from each cell transfected with pCHR012, pCHR042, pCHR067, pCHR068, pCHR069 and pELC2+HC. 図９は、pELC2+HC、pCHR042、pCHR069を導入した各細胞から分泌されたハーセプチン量のグラフを示している。FIG. 9 shows a graph of the amount of Herceptin secreted from each cell transfected with pELC2+HC, pCHR042, and pCHR069.
　以下、本発明の実施形態について説明する。以下の実施形態は、例示であって、本発明の範囲は、以下の実施形態で示すものに限定されない。なお、同様な内容については繰り返しの煩雑をさけるために、摘示説明を省略する。 Embodiments of the present invention will be described below. The following embodiments are examples, and the scope of the present invention is not limited to those shown in the following embodiments. In addition, in order to avoid complication of repetition about the same content, the brief description is abbreviate|omitted.
定義
　便宜上、本願で使用される特定の用語は、ここに集めている。別途規定されない限り、本願で使用される全ての技術用語及び科学用語は、本発明が属する技術分野の当業者が一般的に理解するのと同じ意味を有する。文脈で別途明記されない限り、単数形「a」、「an」及び「the」は複数の言及を含む。 Definitions For convenience, certain terms used in this application are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless the context clearly dictates otherwise, the singular forms "a,""an," and "the" include plural references.
　本発明で示す数値範囲及びパラメーターは、近似値であるが、特定の実施例に示されている数値は可能な限り正確に記載している。しかしながら、いずれの数値も本質的に、それぞれの試験測定値に見られる標準偏差から必然的に生じる特定の誤差を含んでいる。また、本明細書で使用する「約」という用語は、一般に、所与の値又は範囲の10％、5％、1％又は0.5％以内を意味する。或いは、用語「約」は、当業者が考慮する場合、許容可能な標準誤差内にあることを意味する。 Numerical ranges and parameters shown in the present invention are approximate values, but numerical values shown in specific examples are described as accurately as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error, as considered by one of ordinary skill in the art.
　本明細書、図面及び配列表の塩基配列は、別途規定されない限り、5'末端から3'末端の順番で記載している。例えば、「100番目の塩基」は、5'末端から100番目の塩基を意味する。用語「逆相補配列」は、ある配列に対して逆相補の関係にある配列を意味する。例えば、5'-AAATTCGG-3'配列の逆相補配列は、5'-CCGAATTT-3'である。また、「上流」は、5'末端方向を指し、「下流」は、3'末端方向を意味する。 Unless otherwise specified, the nucleotide sequences in this specification, drawings and sequence listing are listed in order from the 5' end to the 3' end. For example, "100th base" means the 100th base from the 5' end. The term "reverse complementary sequence" means a sequence that is in a reverse complementary relationship to a given sequence. For example, the reverse complement of the 5'-AAATTCGG-3' sequence is 5'-CCGAATTT-3'. Also, "upstream" refers to the direction of the 5' end, and "downstream" means the direction of the 3' end.
　開始コドンは、タンパク質の合成開始を指定するコドンを指し、通常は、塩基配列ATG又はAUGで表される。 The initiation codon refers to a codon that specifies the initiation of protein synthesis, and is usually represented by the nucleotide sequence ATG or AUG.
　本実施形態にかかる核酸分子(例えば、DNA、領域、カセット、ゲノム及びベクタープラスミド)の塩基配列は、その塩基配列と、
1）９５％以上の配列相同性を有する塩基配列である、または、
2）１または数塩基が欠失、置換、付加されている塩基配列である、または、
3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列であっても良い。 The nucleotide sequence of the nucleic acid molecule (e.g., DNA, region, cassette, genome, and vector plasmid) according to this embodiment includes the nucleotide sequence,
1) is a nucleotide sequence with 95% or more sequence homology, or
2) a nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
3) It may be a nucleotide sequence that can hybridize with a complementary sequence under stringent conditions.
　なお、本明細書において同一性・相同性を示す値は、BLASTなどの公知のプログラムを利用することにより算出することができる。配列番号に示される塩基配列と配列相同性を有する塩基配列は、９５％以上の配列相同性を有していればよく、例えば、９６％以上、９７％以上、９８％以上、９９％以上、９９．５％以上、９９．８％以上であっても良い。 The value indicating identity/homology in this specification can be calculated using a known program such as BLAST. A nucleotide sequence having sequence homology with the nucleotide sequence shown in the SEQ ID NO may have a sequence homology of 95% or more, for example, 96% or more, 97% or more, 98% or more, 99% or more, It may be 99.5% or more, or 99.8% or more.
　本明細書において、「１または数塩基が欠失、置換、付加されている塩基配列」における「数塩基」は、例えば、２～１０塩基、２～９塩基、２～８塩基、２～７塩基、２～６塩基、２～５塩基、２～４塩基、２～３塩基、２塩基である。欠失、置換、付加した塩基の数は、一般的に少ないほど好ましい。塩基の欠失、置換、付加のうち２種以上が同時に生じてもよい。なお、塩基の欠失、置換、付加については、公知の技術を用いることができる。 As used herein, "several bases" in "a base sequence in which one or several bases are deleted, substituted, or added" are, for example, 2 to 10 bases, 2 to 9 bases, 2 to 8 bases, 2 to 7 base, 2-6 bases, 2-5 bases, 2-4 bases, 2-3 bases, and 2 bases. The number of deleted, substituted or added bases is generally preferably as small as possible. Two or more of deletion, substitution, and addition of bases may occur simultaneously. In addition, well-known techniques can be used for deletion, substitution, and addition of bases.
　本明細書において、「ストリンジェントな条件」とは、例えば以下の（１）（２）などが挙げられる。
（１）イオン強度が低く洗浄温度が高い状態として、たとえば０．０１５ＭのＮａＣｌ／０．００１５Ｍのクエン酸ナトリウム（チトラート）／０．１％ＳＤＳで温度５０℃が挙げられる。あるいは
（２）ハイブリッド形成中にホルムアミド等の変性剤を使用して、たとえば、５０％（vol/vol）ホルムアミドに、０．１％ウシ血清アルブミン／０．１％Ｆｉｃｏｌｌ／０．１％polyinylpyrrolidone／５０ｍＭリン酸ナトリウムバッファｐＨ６．５、７５０ｍＭＮａＣｌ、７５ｍＭクエン酸ナトリウムで温度４２℃とすることが挙げられる。別の例としては、５０％ホルムアミド、５×ＳＳＣ（０．７５Ｍ ＮａＣｌ，０．０７５Ｍクエン酸ナトリウム），５０ｍＭ リン酸ナトリウム（ｐＨ６．８）， ０．１％ピロリン酸ナトリウム, ５×デンハルト液、超音波処理されたサーモンスパームＤＮＡ（５０μｇ／ｍｌ），０．１％ＳＤＳ，１０％硫酸デキストラン，温度４２度，洗浄温度４２℃，０．２×ＳＳＣ，0.1％ＳＤＳが挙げられる。
また、当業者であれば、クリアで検出可能なハイブリダイゼーションシグナルを得るべく、ストリンジェントな条件を適宜変更できることは、明らかである。 As used herein, "stringent conditions" include, for example, the following (1) and (2).
(1) Low ionic strength and high washing temperature include, for example, 0.015 M NaCl/0.0015 M sodium citrate (titrate)/0.1% SDS at a temperature of 50°C. or (2) using a denaturing agent such as formamide during hybridization, e.g., 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyinylpyrrolidone/ 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at a temperature of 42°C. Another example is 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, Sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, 10% dextran sulfate, temperature 42° C., washing temperature 42° C., 0.2×SSC, 0.1% SDS.
It will also be appreciated by those of ordinary skill in the art that stringent conditions can be altered as appropriate to obtain a clear and detectable hybridization signal.
　なお、本明細書において、分子生物学の手法（例えば、クローニング、プラスミド抽出、DNA断片の切断、連結、ハイブリダイゼーション、部位特異的変異導入法、PCR法、ウエスタンブロット法等々の手法）は、当業者によく知られている通常の方法を採用することができる。これらの方法は、 Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition",Cold Spring Harbor Laboratory Press, (1989)等を参照することができる。 As used herein, molecular biology techniques (e.g., cloning, plasmid extraction, DNA fragment cleavage, ligation, hybridization, site-directed mutagenesis, PCR, Western blotting, etc.) Conventional methods well known to those skilled in the art can be employed. For these methods, see Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, (1989), etc. .
実施形態
選択マーカー発現カセット
　本実施形態にかかる選択マーカー発現カセットは、
　発現調節配列DNAを備え、
　上記発現調節配列DNAは、１又は複数の副発現調節配列DNAを備え、
　上記副発現調節配列DNAは、改変型Kozak配列DNAと上記改変型Kozak配列DNAの下流に上流ORF配列DNAとを備え、
　上記副発現調節配列DNAの塩基配列は、開始コドンの塩基配列を１つだけ備える。 Embodiment Selectable Marker Expression Cassette The selectable marker expression cassette according to this embodiment is
comprising an expression control sequence DNA,
The expression control sequence DNA comprises one or more secondary expression control sequence DNAs,
The secondary expression regulatory sequence DNA comprises a modified Kozak sequence DNA and an upstream ORF sequence DNA downstream of the modified Kozak sequence DNA,
The base sequence of the secondary expression regulatory sequence DNA has only one start codon base sequence.
　Kozak配列DNAの塩基配列は、真核生物のmRNAに出現する共通配列であり、主に翻訳の開始に関与している。ただし厳密な共通配列ではなく、不一致のあることも非常に多い。脊椎動物のKozak配列は、(gcc)gccRccAUGG（配列番号：３９）と表され、なかでも開始コドン(AUG)の3塩基上流のR（プリン塩基、アデニン又はグアニン）と開始コドンの次のGが重要な役割を果たすと考えられている（Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987;15(20):8125-8148. doi:10.1093/nar/15.20.8125）。Kozak配列において、（１）小文字は、塩基が変わり得るが、その位置でのもっとも一般的な塩基を表示し、（２）「AUGG」配列は、高度に保存された塩基を指し示し、（３）括弧中の配列(gcc)は不確かな意義(uncertain significance)のものである。 The base sequence of Kozak sequence DNA is a common sequence that appears in eukaryotic mRNA and is mainly involved in the initiation of translation. However, it is not a strict consensus sequence and very often there are discrepancies. The Kozak sequence of vertebrates is represented by (gcc)gccRccAUGG (SEQ ID NO: 39). considered to play an important role (Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987;15(20):8125-8148. doi:10.1093/nar/15.20 .8125). In Kozak sequences, (1) lower case letters indicate the most common base at that position, although bases may vary, (2) "AUGG" sequences indicate highly conserved bases, and (3) Sequences in brackets (gcc) are of uncertain significance.
　本実施形態における改変型Kozak配列DNAは、Kozak配列DNAとは異なる塩基配列を有する。上記改変型Kozak配列DNAは、配列番号：３で表される塩基配列（GCCACC）、又は、その変異型塩基配列
（その塩基配列と、
1）９５％以上の配列相同性を有する塩基配列、又は、
2）１または数塩基が欠失、置換、付加されている塩基配列、又は、
3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列）
からなるであってもよい。
　上記改変型Kozak配列DNAの塩基配列には、開始コドンとしての役割を果たすATG配列が含まれない。 The modified Kozak sequence DNA in this embodiment has a base sequence different from that of the Kozak sequence DNA. The above-mentioned modified Kozak sequence DNA has a base sequence represented by SEQ ID NO: 3 (GCCACC), or a mutant base sequence thereof (the base sequence and
1) A nucleotide sequence with 95% or more sequence homology, or
2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
3) A nucleotide sequence that can hybridize with a complementary sequence under stringent conditions)
It may consist of
The nucleotide sequence of the modified Kozak sequence DNA does not contain an ATG sequence that serves as an initiation codon.
　本実施形態における上流ORF配列DNAは、改変型Kozak配列DNAの下流に位置する。上流ORF配列DNAは、開始コドン（ATG）を１つだけ備える。言い換えれば、副発現調節配列DNAは、開始コドンを１つだけ備える。ある実施形態において、上流ORF配列DNAの開始コドン（ATG）は、その5'末端に位置する。上流ORF配列DNAの塩基配列は、開始コドン（ATG）の下流に、上流ORFの開始コドンとしての役割を果たさないATG配列を備えていてもよい。 The upstream ORF sequence DNA in this embodiment is located downstream of the modified Kozak sequence DNA. The upstream ORF sequence DNA has only one initiation codon (ATG). In other words, the secondary expression control sequence DNA has only one initiation codon. In certain embodiments, the initiation codon (ATG) of the upstream ORF sequence DNA is located at its 5' end. The nucleotide sequence of the upstream ORF sequence DNA may have an ATG sequence downstream of the initiation codon (ATG) that does not serve as an initiation codon for the upstream ORF.
　上記上流ORF配列DNAは、配列番号：１（ATGGCTTGA）又は配列番号：２（ATGGCTCTAGAAATCGGATGA）で表される塩基配列、又は、その変異型塩基配列
（その塩基配列と、
1）９５％以上の配列相同性を有する塩基配列、又は、
2）１または数塩基が欠失、置換、付加されている塩基配列、又は、
3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列）
からなるであってもよい。
　上流ORF配列DNAの塩基配列には、配列番号：３で表される塩基配列もその変異型塩基配列も含まれない。 The upstream ORF sequence DNA has a base sequence represented by SEQ ID NO: 1 (ATGGCTTGA) or SEQ ID NO: 2 (ATGGCTCTAGAAATCGGATGA), or a variant base sequence thereof (the base sequence and
1) A nucleotide sequence with 95% or more sequence homology, or
2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
3) A nucleotide sequence that can hybridize with a complementary sequence under stringent conditions)
It may consist of
The base sequence of the upstream ORF sequence DNA does not include the base sequence represented by SEQ ID NO: 3 or its mutant base sequence.
　ある実施形態において、上流ORF配列DNAは、改変型Kozak配列DNAに直接連結している（言い換えれば、いかなる塩基も上流ORF配列DNAと改変型Kozak配列DNAとの間に存在しない）。別の実施形態において、上流ORF配列DNAは、スペーサーDNAを介して改変型Kozak配列DNAに連結している。スペーサーDNAは、１、２、３、４又は５つの任意の塩基からなっていてもよい。スペーサーDNAは、開始コドンとしての役割を果たすATG配列を備えない。 In certain embodiments, the upstream ORF sequence DNA is directly linked to the modified Kozak sequence DNA (in other words, no bases are present between the upstream ORF sequence DNA and the modified Kozak sequence DNA). In another embodiment, the upstream ORF sequence DNA is linked to the modified Kozak sequence DNA via spacer DNA. The spacer DNA may consist of 1, 2, 3, 4 or 5 arbitrary bases. Spacer DNA does not have an ATG sequence that serves as an initiation codon.
　選択マーカー発現カセットは、発現調節配列DNAに加えて、プロモーター（例えば、マウスPGKプロモーター）及び/又はポリAシグナル（例えば、BGHポリAシグナル）を備えていてもよい。また、選択マーカー発現カセットは、蛍光選択マーカー遺伝子（例：緑色蛍光タンパク質（GFP）をコードするDNA）、薬剤選択マーカー遺伝子（例えば、ピューロマイシン耐性遺伝子及びネオマイシン耐性遺伝子））及び/又は栄養選択マーカー遺伝子（例：グルタミン合成酵素遺伝子）を含む選択マーカー遺伝子を備えていてもよい。ある実施形態において、選択マーカー発現カセットは、プロモーター、発現調節配列DNA、選択マーカー遺伝子及びポリAシグナルを備えていてもよい。 The selectable marker expression cassette may comprise a promoter (eg mouse PGK promoter) and/or a poly A signal (eg BGH poly A signal) in addition to the expression control sequence DNA. In addition, the selection marker expression cassette includes a fluorescent selection marker gene (e.g., DNA encoding green fluorescent protein (GFP)), a drug selection marker gene (e.g., puromycin resistance gene and neomycin resistance gene), and/or a nutritional selection marker. Selectable marker genes including genes (eg, glutamine synthetase gene) may be provided. In certain embodiments, the selectable marker expression cassette may comprise a promoter, expression control sequence DNA, selectable marker gene and poly A signal.
　「選択マーカー遺伝子」の発現量は、本実施形態の構成を採用していない（発現抑制を受けていない）「選択マーカー遺伝子」の発現量と比較して、０．５％、１％、５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、９５％又は９９％以上減弱している。 The expression level of the "selective marker gene" is 0.5%, 1%, 5% compared to the expression level of the "selective marker gene" that does not employ the configuration of this embodiment (not subjected to expression suppression). %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more.
　発現調節配列DNAは、１又は複数の副発現調節配列DNAを備える。ある実施形態において、発現調節配列DNAが複数の副発現調節配列DNAを備える場合、ある副発現調節配列DNAは、隣接する副発現調節配列DNAと直接接続している。別の実施形態において、発現調節配列DNAが複数の副発現調節配列DNAを備える場合、ある副発現調節配列DNAは、スペーサーDNAを介して隣接する副発現調節配列DNAと直接接続している。スペーサーDNAは、１、２、３、４又は５つの任意の塩基からなっていてもよい。スペーサーDNAは、開始コドンとしての役割を果たすATG配列を備えない。ある実施形態において、１又は複数の副発現調節配列DNAは、１つ、２つ又は３つの副発現調節配列DNAである。 The expression control sequence DNA comprises one or more secondary expression control sequence DNAs. In certain embodiments, when the expression control sequence DNA comprises multiple secondary expression control sequence DNAs, one secondary expression control sequence DNA is directly connected to adjacent secondary expression control sequence DNAs. In another embodiment, when the expression control sequence DNA comprises a plurality of secondary expression control sequence DNAs, one secondary expression control sequence DNA is directly connected to adjacent secondary expression control sequence DNAs via spacer DNAs. The spacer DNA may consist of 1, 2, 3, 4 or 5 arbitrary bases. Spacer DNA does not have an ATG sequence that serves as an initiation codon. In certain embodiments, the one or more secondary expression control sequence DNAs are 1, 2 or 3 secondary expression control sequence DNAs.
　上記上流ORF配列DNAの塩基配列が配列番号：２からなる場合、発現調節配列DNAは、１つの副発現調節配列DNAを備えていてもよい。上記上流ORF配列DNAの塩基配列が配列番号：２からなる場合、上記選択マーカー遺伝子の開始コドン配列は、上記上流ORF配列DNAの塩基配列中に含まれて（存在して）いてもよい。また、上記上流ORF配列DNAの塩基配列が配列番号：２からなる場合、上記上流ORF配列配列DNAの読み枠は、上記選択マーカー遺伝子の開始コドンとオーバーラップしてもよい。 When the base sequence of the upstream ORF sequence DNA consists of SEQ ID NO: 2, the expression control sequence DNA may comprise one secondary expression control sequence DNA. When the nucleotide sequence of the upstream ORF sequence DNA consists of SEQ ID NO: 2, the initiation codon sequence of the selectable marker gene may be included (present) in the nucleotide sequence of the upstream ORF sequence DNA. Moreover, when the nucleotide sequence of the upstream ORF sequence DNA consists of SEQ ID NO: 2, the reading frame of the upstream ORF sequence DNA may overlap the start codon of the selectable marker gene.
　Kozak配列、改変型Kozak配列及びそれらの変異型塩基配列は、選択マーカー遺伝子と隣接する上流ORF配列の間には存在しない。 The Kozak sequence, modified Kozak sequence and their mutant base sequences are not present between the selectable marker gene and the adjacent upstream ORF sequence.
発現ベクター
　本実施形態にかかる発現ベクターは、選択マーカー発現カセットを備える。 Expression Vector The expression vector according to this embodiment comprises a selectable marker expression cassette.
　本実施形態における発現ベクターは、クローニングベクターに、選択マーカー発現カセットを挿入等することで作成することができる。上記のクローニングベクターとしては、例えば、プラスミド、ファージ、ファージミド、コスミド、フォスミドまたは、人工染色体などがあげられる。具体的には大腸菌由来のプラスミド（例えばｐＢＲ３２２、ｐＢＲ３２５、ｐＵＣ１２、ｐＵＣ１３）、酵母由来プラスミド（例えばｐＳＨ１９、ｐＳＨ１５）、枯草菌由来のプラスミド（例えばｐＵＢ１１０、ｐＴＰ５、ｐＣ１９４）、動物細胞発現プラスミド（例えばｐＡ１－１１、ｐＸＴ１、ｐＲｃ／ＣＭＶ、ｐＲｃ／ＲＳＶ、ｐｃＤＮＡＩ／Ｎｅｏ）、λファージなどのバクテリオファージ、ＨＩＶ、アデノウイルス、レトロウイルス、ワクシニアウイルス、バキュロウイルスなどのウイルス由来のベクターや人工染色体などを用いることができる。 The expression vector in this embodiment can be created by inserting a selection marker expression cassette into a cloning vector. Examples of the above cloning vectors include plasmids, phages, phagemids, cosmids, fosmids, artificial chromosomes, and the like. Specifically, E. coli-derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13), yeast-derived plasmids (e.g., pSH19, pSH15), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, pC194), animal cell expression plasmids (e.g., pA1 -11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo), bacteriophages such as λ phage, viral vectors such as HIV, adenovirus, retrovirus, vaccinia virus, baculovirus, and artificial chromosomes be able to.
　本実施形態にかかる発現ベクターは、２つの末端tDNAインシュレーターを更に備えていてもよい。本実施形態にかかる発現ベクターが２つの末端tDNAインシュレーターを備えている場合、上記選択マーカー発現カセットは、上記２つの末端tDNAインシュレーターの間に位置することが好ましい。末端tDNAインシュレーターは、プロモーター干渉、サイレンシングを低減させ、遺伝子発現安定化に関与する、遺伝子の発現単位を区切ることが可能な塩基配列からなるDNAであり、例えば、マウスtRNA遺伝子の塩基配列からなるDNAを挙げることができる。 The expression vector according to this embodiment may further comprise two terminal tDNA insulators. When the expression vector according to this embodiment comprises two terminal tDNA insulators, the selection marker expression cassette is preferably positioned between the two terminal tDNA insulators. A terminal tDNA insulator is a DNA consisting of nucleotide sequences capable of delimiting expression units of a gene, which reduces promoter interference, silencing, and is involved in stabilizing gene expression, for example, consisting of the nucleotide sequence of the mouse tRNA gene. DNA can be mentioned.
　本実施形態にかかる発現ベクターは、１又は複数の目的タンパク質発現カセットを備えることができる。本実施形態にかかる発現ベクターが１又は複数の目的タンパク質発現カセットを備える場合、上記１又は複数の目的タンパク質発現カセットは、上記２つの末端tDNAインシュレーターの間に位置することが好ましい。目的タンパク質発現カセットは、目的タンパク質を発現する遺伝子を備える。一実施形態において、目的タンパク質発現カセットは、目的タンパク質を発現する遺伝子に加えて、プロモーター（例えば、ヒトEF1αプロモーター）、翻訳効率を高める因子（例えば、βグロビンリーダー配列）、mRNAを安定化する因子（例えば、Woodchuck hepatitis virus Post-transcriptional Regulatory Element（WPRE）配列及びポリAシグナル（SV40ポリAシグナル）からなる群より選択される少なくとも１つを備えていてもよい。 The expression vector according to this embodiment can comprise one or more target protein expression cassettes. When the expression vector according to this embodiment comprises one or more protein-of-interest expression cassettes, the one or more protein-of-interest expression cassettes are preferably located between the two terminal tDNA insulators. A protein of interest expression cassette comprises a gene that expresses the protein of interest. In one embodiment, the target protein expression cassette includes, in addition to the gene expressing the target protein, (For example, it may have at least one selected from the group consisting of a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE) sequence and a poly A signal (SV40 poly A signal).
　「目的タンパク質」は、「選択マーカー遺伝子」の発現量を減弱化させることで、その発現量を増加させたいタンパク質を指す。本実施形態の構成中における「目的タンパク質」の発現量は、本発明の構成を採用していない（選択マーカーが発現抑制を受けていない）「目的タンパク質」の発現量と比較して、１．５倍、２．０倍、２．５倍、３．０倍、３．５倍、４．０倍、４．５倍又は５．０倍以上増加する。 "Target protein" refers to a protein whose expression level is to be increased by attenuating the expression level of the "selection marker gene". The expression level of the "target protein" in the configuration of this embodiment is compared with the expression level of the "target protein" that does not employ the configuration of the present invention (the selection marker is not suppressed in expression). 5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold or more than 5.0-fold.
　本実施形態にかかる発現ベクターは、１又は複数の中間tDNAインシュレーターを備えることができる。本実施形態にかかる発現ベクターが複数の目的タンパク質発現カセットを備える場合、少なくとも１の上記中間tDNAインシュレーターが、上記複数の目的タンパク質発現カセットのうちの１つと隣接する目的タンパク質発現カセットとの間に存在することが好ましい。中間tDNAインシュレーターは、中間tDNAインシュレーターと同じであってもよく、例えば、マウスtRNA遺伝子の塩基配列からなるDNAを挙げることができる。 The expression vector according to this embodiment can comprise one or more intermediate tDNA insulators. When the expression vector according to this embodiment comprises a plurality of target protein expression cassettes, at least one intermediate tDNA insulator is present between one of the plurality of target protein expression cassettes and the adjacent target protein expression cassette. preferably. The intermediate tDNA insulator may be the same as the intermediate tDNA insulator, and may include, for example, DNA consisting of the base sequence of the mouse tRNA gene.
実施例１
発現ベクター作製用プラスミド（pCHR002）および第2遺伝子クローニング用プラスミド（pCHR006）の構築
　VectorBuilder社のオンラインプラットフォーム上で利用可能な標準コンポーネントで構成され、VectorBuilder社によって構築されたプラスミド（ベクターID: VB900085-9593ncv及び VB190621-1088bqm）および人工合成したtDNAを含むプラスミド（Ebersole T, Kim JH, Samoshkin A, et al. tRNA genes protect a reporter gene from epigenetic silencing in mouse cells. Cell Cycle. 2011;10(16):2779-2791.を参照）を用意した。これらのプラスミドに基づいて、図1に表示する制限酵素サイトが付加されるように表１に示すコンポーネントをPCR法によって増幅した。 Example 1
Construction of expression vector construction plasmid (pCHR002) and second gene cloning plasmid (pCHR006) Plasmid constructed by VectorBuilder, consisting of standard components available on VectorBuilder's online platform (Vector ID: VB900085-9593ncv and VB190621-1088bqm) and a plasmid containing artificially synthesized tDNA (Ebersole T, Kim JH, Samoshkin A, et al. tRNA genes protect a reporter gene from epigenetic silencing in mouse cells. Cell Cycle. 2011;10(16):2779 -2791.) was prepared. Based on these plasmids, the components shown in Table 1 were amplified by PCR so that the restriction enzyme sites shown in FIG. 1 were added.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　各コンポーネントは、表２に示すプライマーを使用してPCR法によって増幅させた。 Each component was amplified by PCR using the primers shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　これらのコンポーネントをNEBuilder HiFi DNA Assembly（New England Biolabs）を用いて結合させた。結合DNA断片は、AscI_tDNA-F2（配列番号：18）とNotI_tDNA-R2（配列番号：19）のプライマーセットを用いてPCRによって増幅させた。増幅させた結合DNA断片を、VectorBuilder社によって構築されたプラスミド(VB900085-9593ncv)骨格の制限酵素AscI、NotIサイトへ導入し、新しい発現ベクター作製用プラスミドpCHR002を構築した（図1）。pCHR002は、tDNA、ヒトEF-1αpromoter、SV40 late pA、mPGK promoter、PuroR、BGH pA、細菌複製起点（pUC ori）、アンピシリン耐性マーカー（AmpR）を含有する。 These components were combined using NEBuilder HiFi DNA Assembly (New England Biolabs). The bound DNA fragment was amplified by PCR using a primer set of AscI_tDNA-F2 (SEQ ID NO: 18) and NotI_tDNA-R2 (SEQ ID NO: 19). The amplified ligated DNA fragment was introduced into the restriction enzymes AscI and NotI sites of the backbone of a plasmid (VB900085-9593ncv) constructed by VectorBuilder to construct a new expression vector construction plasmid pCHR002 (Fig. 1). pCHR002 contains tDNA, human EF-1α promoter, SV40 late pA, mPGK promoter, PuroR, BGH pA, bacterial origin of replication (pUC ori), ampicillin resistance marker (AmpR).
　同様に、tDNA、ヒトEF-1αpromoter、SV40 late pA、pUC ori、AmpRを含有する、第2遺伝子クローニング用プラスミドpCHR006を構築した（図２）。各コンポーネントは、表３に示すプライマーを使用してPCR法によって増幅させた。 Similarly, a second gene cloning plasmid pCHR006 containing tDNA, human EF-1α promoter, SV40 late pA, pUC ori, and AmpR was constructed (Fig. 2). Each component was amplified by PCR using the primers shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　これらのコンポーネントをNEBuilder HiFi DNA Assembly（New England Biolabs）を用いて結合させた。結合DNA断片は、AscI_MluI_NdeI_tDNA-F（配列番号：20）とNotI_PacI_EcoT22I_SV40pA-R（配列番号：21）のプライマーセットを用いてPCRによって増幅させた。増幅させた結合DNA断片を、VectorBuilder社によって構築されたプラスミド(VB900085-9593ncv)骨格の制限酵素AscI、NotIサイトへ導入し、pCHR006を構築した。 These components were combined using NEBuilder HiFi DNA Assembly (New England Biolabs). The bound DNA fragment was amplified by PCR using the primer set AscI_MluI_NdeI_tDNA-F (SEQ ID NO: 20) and NotI_PacI_EcoT22I_SV40pA-R (SEQ ID NO: 21). The amplified ligated DNA fragment was introduced into the restriction enzyme AscI and NotI sites of the backbone of a plasmid (VB900085-9593ncv) constructed by VectorBuilder to construct pCHR006.
ハーセプチン発現ベクターの構築
　人工合成したハーセプチン遺伝子を含むプラスミド（pELC2+HC（TOYOBO, Mammalian PowerExpress System（登録商標））に基づいて、ハーセプチン軽鎖遺伝子（配列番号：33）及びハーセプチン重鎖遺伝子（配列番号：34）をPCRによって増幅した。使用したプライマーは表４に示す。 Construction of Herceptin Expression Vector Herceptin light chain gene (SEQ ID NO: 33) and Herceptin heavy chain gene (SEQ ID NO: :34) was amplified by PCR, and the primers used are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　ハーセプチン軽鎖遺伝子をHindIII及びSalI制限酵素サイトを付加するようにPCRによって増幅した。増幅したハーセプチン軽鎖遺伝子をpCHR002のHindIII及びSalIサイトへ導入することで、pCHR002-Lcを構築した（図3）。同様に、ハーセプチン重鎖遺伝子をHindIII及びSbfI制限酵素サイトを付加するようにPCRによって増幅した。増幅したハーセプチン重鎖遺伝子をpCHR006のHindIII及びSbfIサイトへ導入することで、pCHR008を構築した（図4）。次に、pCHR008からハーセプチン重鎖遺伝子を含む断片を制限酵素MluI及びNsiIによって切り出した。切り出した断片をpCHR002-LcのMluI及びNsiIサイトへ導入することで、ハーセプチン発現ベクターpCHR012を構築した（図5）。 The Herceptin light chain gene was amplified by PCR to add HindIII and SalI restriction enzyme sites. pCHR002-Lc was constructed by introducing the amplified Herceptin light chain gene into the HindIII and SalI sites of pCHR002 (Fig. 3). Similarly, the Herceptin heavy chain gene was amplified by PCR to add HindIII and SbfI restriction enzyme sites. pCHR008 was constructed by introducing the amplified Herceptin heavy chain gene into the HindIII and SbfI sites of pCHR006 (Fig. 4). Next, a fragment containing the Herceptin heavy chain gene was excised from pCHR008 with restriction enzymes MluI and NsiI. Herceptin expression vector pCHR012 was constructed by introducing the excised fragment into the MluI and NsiI sites of pCHR002-Lc (Fig. 5).
マーカー減弱化ハーセプチン発現ベクター（pCHR027、pCHR042、pCHR067、pCHR068、pCHR069）の構築
　pCHR012を鋳型にPacI-YB_TATA-B-X-ΔK-PuroR-F（配列番号：26）とBamHI_PuroR-R（配列番号：13）のプライマーセットで増幅したPCR産物を、pCHR012のXbaI及びBamHIサイトへ導入した。これによって、ピューロマイシン耐性遺伝子のコザック配列に変異を入れたpCHR027を構築した（図6）。 Construction of marker-attenuated Herceptin expression vectors (pCHR027, pCHR042, pCHR067, pCHR068, pCHR069) PacI-YB_TATA-BX-ΔK-PuroR-F (SEQ ID NO: 26) and BamHI_PuroR-R (SEQ ID NO: 13) using pCHR012 as a template was introduced into the XbaI and BamHI sites of pCHR012. This resulted in construction of pCHR027 in which the Kozak sequence of the puromycin resistance gene was mutated (Fig. 6).
　pCHR027を鋳型にPacI_mPGKpro-F（配列番号：10）とXbaI-K-uORF-mPGK-R（配列番号：35）のプライマーセットで増幅したPCR産物を、pCHR027のPacI及びXbaIサイトへ導入した。これによって、ピューロマイシン耐性遺伝子に上流ORFを挿入したpCHR042を構築した（図6）。同様に、表５に示すプラスミドを、対応するプライマーセットを用いて増幅させたPCR産物をpCHR027のPacI及びXbaIサイトへ導入することで作成した。 Using pCHR027 as a template, a PCR product amplified with a primer set of PacI_mPGKpro-F (SEQ ID NO: 10) and XbaI-K-uORF-mPGK-R (SEQ ID NO: 35) was introduced into the PacI and XbaI sites of pCHR027. This resulted in the construction of pCHR042 with an upstream ORF inserted into the puromycin resistance gene (Fig. 6). Similarly, the plasmids shown in Table 5 were constructed by introducing the PCR products amplified using the corresponding primer sets into the PacI and XbaI sites of pCHR027.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　図6に示す通り、pCHR067は、ピューロマイシン耐性遺伝子に上流ORFが2つ挿入され、pCHR068は、ピューロマイシン耐性遺伝子に上流ORFが3つ挿入され、pCHR069は、ピューロマイシン耐性遺伝子の開始コドンにオーバーラップするように上流ORFが挿入されている。 As shown in Figure 6, pCHR067 has two upstream ORFs inserted into the puromycin resistance gene, pCHR068 has three upstream ORFs inserted into the puromycin resistance gene, and pCHR069 has an insertion over the start codon of the puromycin resistance gene. Upstream ORF is inserted to wrap.
pCHR012、pCHR042、pCHR067、pCHR068、pCHR069、pELC2+HCの細胞導入
　浮遊培養に馴化させた3×105 cells/mLのチャイニーズハムスター卵巣由来細胞CHO-G1（株式会社chromocenterが樹立）を12ウェルプレートに1 mLずつ播種した。一晩接着培養して、トランスフェクション用のCHO-G1細胞を準備した。5% FBSを加えたProCHO4培地（HT Supplement (1×)（GIBCO製）、L-Glutamine（2 mM）（GIBCO製）、Penicillin-Streptomycin (100 U/mL)（GIBCO製）を添加したProCHO4 Protein-free CHO Medium（LONZA製））を培地として使用した。 Cell transfer of pCHR012, pCHR042, pCHR067, pCHR068, pCHR069, and pELC2+HC 3×10 5 cells/mL Chinese hamster ovary-derived cells CHO-G1 (established by chromocenter, Inc.) adapted to suspension culture were placed in a 12-well plate. 1 mL each was seeded. CHO-G1 cells were prepared for transfection by overnight adherent culture. ProCHO4 medium with 5% FBS (HT Supplement (1×) (GIBCO), L-Glutamine (2 mM) (GIBCO), ProCHO4 Protein supplemented with Penicillin-Streptomycin (100 U/mL) (GIBCO) -free CHO Medium (manufactured by LONZA) was used as the medium.
　プラスミドとしてpCHR012、pCHR042、pCHR067、pCHR068、pCHR069、pELC2+HC（図7、TOYOBO, Mammalian PowerExpress System（登録商標）に人工合成したハーセプチン遺伝子を導入したハーセプチン発現ベクター）を使用した。トランスフェクションは、以下の方法で行った。1.6 μgのプラスミドベクターを100 μL Opti-MEM I Reduced Serum Medium（GIBCO製）で希釈した。4 μL Lipofectamine 2000 Transfection Reagent (Invitrogen製)を100 μL Opti-MEM I Reduced Serum Medium（GIBCO製）で希釈した。5分間放置した後、プラスミドの希釈液とLipofectamine 2000の希釈液を混合し、20分間放置した。その後、上記CHO-G1細胞（培地をOpti-MEM I Reduced Serum Mediumに置き換えた細胞）に混合液を加え、24時間インキュベートした。インキュベーション後、細胞をAccumax（Innovative Cell Technologies製）で処理して分散させた。分散させた細胞をφ93×19.2 mm細胞培養ディッシュに移し、ProCHO4培地+5% FBSで培養した。翌日、細胞を培養している培地にPuromycin（Sigma-Aldrich製）を7.5 μg/mLの濃度で添加し、6-12日間の薬剤選抜接着培養を行った。選抜培養中、3-4日ごとに培地交換、あるいは適当な細胞密度で継代培養を行った。細胞が十分増殖した時点で5 μg/mL Puromycinを含むProCHO4培地で浮遊静置培養に移行した。浮遊静置培養でさらに1週間程度細胞を培養し、ハーセプチン安定発現CHO-G1ヘテロセルプールを得た。 As plasmids, pCHR012, pCHR042, pCHR067, pCHR068, pCHR069, pELC2+HC (Fig. 7, Herceptin expression vector in which artificially synthesized Herceptin gene was introduced into TOYOBO, Mammalian PowerExpress System (registered trademark)) was used. Transfection was performed by the following method. 1.6 μg of plasmid vector was diluted with 100 μL Opti-MEM I Reduced Serum Medium (manufactured by GIBCO). 4 µL Lipofectamine 2000 Transfection Reagent (manufactured by Invitrogen) was diluted with 100 µL Opti-MEM I Reduced Serum Medium (manufactured by GIBCO). After standing for 5 minutes, the plasmid dilution and the Lipofectamine 2000 dilution were mixed and left for 20 minutes. After that, the mixture was added to the CHO-G1 cells (cells whose medium was replaced with Opti-MEM I Reduced Serum Medium) and incubated for 24 hours. After incubation, the cells were treated with Accumax (Innovative Cell Technologies) and dispersed. The dispersed cells were transferred to a φ93×19.2 mm cell culture dish and cultured in ProCHO4 medium+5% FBS. The next day, Puromycin (manufactured by Sigma-Aldrich) was added to the medium in which the cells were being cultured at a concentration of 7.5 μg/mL, and drug-selective adhesion culture was performed for 6 to 12 days. During the selection culture, the medium was changed every 3-4 days, or subculture was performed at an appropriate cell density. When the cells had grown sufficiently, they were transferred to stationary stationary culture in ProCHO4 medium containing 5 μg/mL Puromycin. The cells were further cultured in floating stationary culture for about one week to obtain a CHO-G1 heterocell pool stably expressing Herceptin.
ハーセプチン生産性の評価
　pCHR012、pCHR042、pCHR067、ppCH068、CHR069、pELC2+HCの各発現ベクターを導入したそれぞれのハーセプチン安定発現CHO-G1ヘテロセルプールを5 μg/mL Puromycinを含む新鮮なProCHO4培地で2×105 cells/mLに調整した。2 mLのセルプールを6ウェルプレートに播種して37℃で浮遊静置培養した。5日後の培養上清を回収し、4000 rpmで5分間遠心して上清を回収することで生産ハーセプチンのサンプルとした。ハーセプチンの定量はAlphaLISA human IgG kit（PerkinElmer製）を用いてEnsight Multimode Plate Reader（PerkinElmer製）で測定した。 Evaluation of Herceptin Productivity Each herceptin stably expressing CHO-G1 heterocell pool transfected with pCHR012, pCHR042, pCHR067, ppCH068, CHR069, and pELC2+HC expression vectors was cultured in fresh ProCHO4 medium containing 5 μg/mL Puromycin. It was adjusted to ×10 5 cells/mL. 2 mL of the cell pool was seeded in a 6-well plate and cultured in suspension at 37°C. After 5 days, the culture supernatant was collected, centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected to obtain a sample of produced Herceptin. Herceptin was quantified with an Ensight Multimode Plate Reader (PerkinElmer) using AlphaLISA human IgG kit (PerkinElmer).
　図8は、pCHR012、pCHR042、pCHR067、pCHR068、pCHR069、pELC2+HCの各発現ベクターを導入したそれぞれのハーセプチン安定発現CHO-G1ヘテロセルプールの培養上清におけるハーセプチン定量の結果を示す。ピューロマイシン耐性遺伝子に上流ORFを挿入したベクター（pCHR042）は、上流ORFを挿入しないベクター（pCHR012）と比較して、ハーセプチン生産量が約3倍に増加した。また、上流ORFを2コピー搭載したベクター(pCHR067)は、ベクター（pCHR042）と比較して、さらにハーセプチン生産量が増加した。同様に、ピューロマイシン耐性遺伝子の開始コドンにオーバーラップするように上流ORFが挿入されているベクター(pCHR069)もベクター(pCHR067)と同程度のハーセプチン生産量であった。 Fig. 8 shows the results of herceptin quantification in the culture supernatant of each herceptin-stably-expressing CHO-G1 heterocell pool into which each expression vector of pCHR012, pCHR042, pCHR067, pCHR068, pCHR069, and pELC2+HC was introduced. The vector (pCHR042) in which the upstream ORF was inserted into the puromycin resistance gene increased herceptin production by about 3-fold compared to the vector (pCHR012) in which the upstream ORF was not inserted. In addition, the vector (pCHR067) loaded with two copies of the upstream ORF further increased herceptin production compared to the vector (pCHR042). Similarly, the vector (pCHR069) in which the upstream ORF was inserted so as to overlap with the initiation codon of the puromycin resistance gene produced Herceptin at the same level as the vector (pCHR067).
実施例２
pELC2+HC、pCHR042、pCHR069のCHO-K1細胞への導入
　CHO-K1細胞（JCRB細胞バンク、IFO50414）を株式会社chromocenterにて浮遊培養に馴化した。上記CHO-G1細胞と同様の方法を用いて、トランスフェクション用のCHO-K1細胞を準備し、pELC2+HC、pCHR042、pCHR069のトランスフェクションを行った。また、上記CHO-G1細胞と同様の方法を用いて、7日間の薬剤選抜接着培養を行い、細胞が十分増殖した時点で5 μg/mL Puromycinを含むProCHO4培地で浮遊静置培養に移行した。浮遊静置培養でさらに2週間程度の細胞培養を行い、ハーセプチン安定発現CHO-K1ヘテロセルプールを得た。 Example 2
Introduction of pELC2+HC, pCHR042, and pCHR069 into CHO-K1 Cells CHO-K1 cells (JCRB cell bank, IFO50414) were adapted to suspension culture at chromocenter. CHO-K1 cells for transfection were prepared and transfected with pELC2+HC, pCHR042, and pCHR069 using the same method as for the CHO-G1 cells described above. In addition, using the same method as the above CHO-G1 cells, drug-selective adhesion culture was performed for 7 days, and when the cells were sufficiently grown, they were transferred to suspension stationary culture in ProCHO4 medium containing 5 μg/mL Puromycin. Cell culture was further carried out for about 2 weeks in stationary stationary culture to obtain a CHO-K1 heterocell pool stably expressing Herceptin.
ハーセプチン生産性の評価
　上記CHO-G1細胞と同様の方法を用いて、pELC2+HC、pCHR042及びCHR069の各発現ベクターを導入したそれぞれのハーセプチン安定発現CHO-K1ヘテロセルプールのハーセプチン生産量を測定した。図9は、pELC2+HC、pCHR042及びpCHR069の各発現ベクターを導入したそれぞれのハーセプチン安定発現CHO-K1ヘテロセルプールの培養上清におけるハーセプチン定量の結果を示す。この結果、pELC2+HCと比較して、pCHR042は約2.8倍、pCHR069は約4.5倍のハーセプチン生産量を示した。 Evaluation of Herceptin Productivity Using the same method as for the above CHO-G1 cells, the herceptin production amount of each of the herceptin stably expressing CHO-K1 heterocell pools introduced with the pELC2+HC, pCHR042 and CHR069 expression vectors was measured. . FIG. 9 shows the results of quantification of herceptin in the culture supernatant of each CHO-K1 heterocell pool stably expressing herceptin into which each expression vector of pELC2+HC, pCHR042 and pCHR069 was introduced. As a result, pCHR042 showed about 2.8-fold higher herceptin production and pCHR069 about 4.5-fold higher than pELC2+HC.

Claims (12)

  1. 　発現調節配列DNAを備え、
    　前記発現調節配列DNAは、１又は複数の副発現調節配列DNAを備え、
    　前記副発現調節配列DNAは、改変型Kozak配列DNAと前記改変型Kozak配列DNAの下流に上流ORF配列DNAとを備え、
    　前記副発現調節配列DNAの塩基配列は、開始コドンの塩基配列を１つだけ備える、
    　選択マーカー発現カセット。     comprising an expression control sequence DNA,
    The expression control sequence DNA comprises one or more secondary expression control sequence DNAs,
    The secondary expression regulatory sequence DNA comprises a modified Kozak sequence DNA and an upstream ORF sequence DNA downstream of the modified Kozak sequence DNA,
    The base sequence of the secondary expression regulatory sequence DNA comprises only one start codon base sequence,
    Selectable marker expression cassette.
  2. 　前記１又は複数の副発現調節配列DNAは、１つ、２つ又は３つの副発現調節配列DNAである、
    　請求項１に記載の選択マーカー発現カセット。     The one or more secondary expression regulatory sequence DNAs are 1, 2 or 3 secondary expression regulatory sequence DNAs,
    The selectable marker expression cassette of claim 1.
  3. 　前記上流ORF配列DNAは、配列番号：１又は２で表される塩基配列、
    又は
    その塩基配列と、
    1）９５％以上の配列相同性を有する塩基配列、又は、
    2）１または数塩基が欠失、置換、付加されている塩基配列、又は、
    3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列
    からなる
    　請求項１又は２に記載の選択マーカー発現カセット。     The upstream ORF sequence DNA has a nucleotide sequence represented by SEQ ID NO: 1 or 2,
    or its base sequence,
    1) A nucleotide sequence with 95% or more sequence homology, or
    2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
    3) The selection marker expression cassette according to claim 1 or 2, comprising a nucleotide sequence capable of hybridizing with a complementary sequence under stringent conditions.
  4. 　前記改変型Kozak配列DNAは、配列番号：３で表される塩基配列、
    又は、
    その塩基配列と、
    1）９５％以上の配列相同性を有する塩基配列、又は、
    2）１または数塩基が欠失、置換、付加されている塩基配列、又は、
    3）相補的な配列とストリンジェントな条件下でハイブリダイズすることができる塩基配列
    からなる
    　請求項１から３のいずれかに記載の選択マーカー発現カセット。     The modified Kozak sequence DNA has a nucleotide sequence represented by SEQ ID NO: 3,
    or
    its base sequence;
    1) A nucleotide sequence with 95% or more sequence homology, or
    2) A nucleotide sequence in which one or several bases have been deleted, substituted, or added, or
    3) The selection marker expression cassette according to any one of claims 1 to 3, comprising a nucleotide sequence capable of hybridizing with a complementary sequence under stringent conditions.
  5. 　請求項１から４のいずれかに記載の選択マーカー発現カセットを備える、
    　発現ベクター。     comprising a selectable marker expression cassette according to any one of claims 1 to 4,
    expression vector.
  6. 　２つの末端tDNAインシュレーターを更に備え、
    　前記選択マーカー発現カセットは、前記２つの末端tDNAインシュレーターの間に位置する、
    　請求項５に記載の発現ベクター。     further comprising two terminal tDNA insulators;
    the selectable marker expression cassette is located between the two terminal tDNA insulators;
    The expression vector according to claim 5.
  7. 　１又は複数の目的タンパク質発現カセットを更に備え、
    　前記１又は複数の目的タンパク質発現カセットは、前記２つの末端tDNAインシュレーターの間に位置する、
    　請求項６に記載の発現ベクター。     further comprising one or more target protein expression cassettes;
    the one or more protein-of-interest expression cassettes are located between the two terminal tDNA insulators;
    The expression vector according to claim 6.
  8. 　更に、１又は複数の中間tDNAインシュレーターを備え、
    　前記１又は複数の目的タンパク質発現カセットは、複数の目的タンパク質発現カセットであり、
    　少なくとも１の前記中間tDNAインシュレーターが、前記複数の目的タンパク質発現カセットのうちの１つと隣接する目的タンパク質発現カセットとの間に存在する、
    　請求項７に記載の発現ベクター。     further comprising one or more intermediate tDNA insulators,
    The one or more protein-of-interest expression cassettes are a plurality of protein-of-interest expression cassettes,
    at least one said intermediate tDNA insulator is present between one of said plurality of protein-of-interest expression cassettes and an adjacent protein-of-interest expression cassette;
    The expression vector according to claim 7.
  9. 　前記中間tDNAインシュレーターは、マウスtRNA遺伝子由来の塩基配列を有する、
    　請求項８に記載の発現ベクター。     The intermediate tDNA insulator has a nucleotide sequence derived from a mouse tRNA gene,
    The expression vector according to claim 8.
  10. 　前記末端tDNAインシュレーターは、マウスtRNA遺伝子由来の塩基配列を有する、
    　請求項６から９に記載の発現ベクター。     The terminal tDNA insulator has a base sequence derived from a mouse tRNA gene,
    The expression vector according to claims 6-9.
  11. 　前記選択マーカー発現カセットは、選択マーカー遺伝子を更に備え、
    　前記選択マーカー遺伝子は、前記発現調節配列DNAの下流に位置する、
    　請求項５から１０のいずれかに記載の発現ベクター。     The selectable marker expression cassette further comprises a selectable marker gene,
    the selectable marker gene is located downstream of the expression control sequence DNA;
    The expression vector according to any one of claims 5-10.
  12. 　前記上流ORF配列DNAの塩基配列が配列番号：２からなる場合、前記選択マーカー遺伝子の開始コドン配列は、前記上流ORF配列DNAの塩基配列中に含まれる、
    　請求項１１に記載の発現ベクター。     When the base sequence of the upstream ORF sequence DNA consists of SEQ ID NO: 2, the start codon sequence of the selectable marker gene is included in the base sequence of the upstream ORF sequence DNA.
    The expression vector according to claim 11.
PCT/JP2022/006667 2021-03-15 2022-02-18 Selection marker expression cassette WO2022196245A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023506894A JP7416355B2 (en) 2021-03-15 2022-02-18 Selectable marker expression cassette

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021041250 2021-03-15
JP2021-041250 2021-03-15

Publications (1)

Publication Number Publication Date
WO2022196245A1 true WO2022196245A1 (en) 2022-09-22

Family

ID=83322300

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/006667 WO2022196245A1 (en) 2021-03-15 2022-02-18 Selection marker expression cassette

Country Status (2)

Country Link
JP (1) JP7416355B2 (en)
WO (1) WO2022196245A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009107775A1 (en) * 2008-02-27 2009-09-03 国立大学法人北海道大学 Expression vector for mass production of foreign gene-derived protein using animal cell and use thereof
US20120301919A1 (en) * 2011-05-24 2012-11-29 Agency For Science, Technology And Research Ires mediated multicistronic vectors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009107775A1 (en) * 2008-02-27 2009-09-03 国立大学法人北海道大学 Expression vector for mass production of foreign gene-derived protein using animal cell and use thereof
US20120301919A1 (en) * 2011-05-24 2012-11-29 Agency For Science, Technology And Research Ires mediated multicistronic vectors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GATHER FABIAN, SCHMITZ KATJA, KOCH KATHRIN, VOGT LEA-MARIE, PAUTZ ANDREA, KLEINERT HARTMUT: "Regulation of human inducible nitric oxide synthase expression by an upstream open reading frame", NITRIC OXIDE: BIOLOGY AND CHEMISTRY., ACADEMIC PRESS, AMSTERDAM, NL, vol. 88, 1 July 2019 (2019-07-01), AMSTERDAM, NL , pages 50 - 60, XP055968245, ISSN: 1089-8603, DOI: 10.1016/j.niox.2019.04.008 *
JOSHUA P FERREIRA, WILLIAM L NODERER, ALEXANDER J DIAZ DE ARCE, CLIFFORD L WANG: "Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation", BIOENGINEERED, LANDES BIOSCIENCE, US, vol. 5, no. 3, 1 May 2014 (2014-05-01), US , pages 186 - 192, XP055539418, ISSN: 2165-5979, DOI: 10.4161/bioe.27607 *
NADERI FATEMEH, HASHEMI MEHRDAD, BAYAT HADI, MOHAMMADIAN OMID, POURMALEKI ES'HAGH, ETEMADZADEH MOHAMMAD HOSSEIN, RAHIMPOUR AZAM: "The Augmenting Effects of the tDNA Insulator on Stable Expression of Monoclonal Antibody in Chinese Hamster Ovary Cells", MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY, vol. 37, no. 5, 1 November 2018 (2018-11-01), pages 200 - 206, XP055968248, DOI: 10.1089/mab.2018.0015 *

Also Published As

Publication number Publication date
JPWO2022196245A1 (en) 2022-09-22
JP7416355B2 (en) 2024-01-17

Similar Documents

Publication Publication Date Title
US8501454B2 (en) Homologous recombination-based DNA cloning compositions
EP3344766B1 (en) Systems and methods for selection of grna targeting strands for cas9 localization
Elango et al. Optimized transfection of mRNA transcribed from ad (A/T) 100 tail-containing vector
EP3684925B1 (en) Non-integrating dna vectors for the genetic modification of cells
EP2890404A1 (en) Dna plasmids with improved expression
US11060108B2 (en) DNA element having the activity of enhancing foreign gene expression
Carnes et al. Critical design criteria for minimal antibiotic‐free plasmid vectors necessary to combine robust RNA Pol II and Pol III‐mediated eukaryotic expression with high bacterial production yields
Radulovich et al. Modified gateway system for double shRNA expression and Cre/lox based gene expression
JP2023062130A (en) Method for selecting cells based on crispr/cas-mediated integration of a detectable tag to target protein
US9012226B2 (en) Bacterial strains with improved plasmid stability
WO2020141124A2 (en) Non-replicative transduction particles with one or more non-native tail fibers and transduction particle-based reporter systems
WO2022196245A1 (en) Selection marker expression cassette
Plumbridge et al. Genes for the two subunits of phenylalanyl-tRNA synthetase of Escherichia coli are transcribed from the same promoter
JP6779513B2 (en) Methods for screening in vivo cloning cell lines, methods for producing in vivo cloning cell lines, cell lines, in vivo cloning methods, and kits for performing in vivo cloning.
KR101346620B1 (en) Novel bacterial lysis protein and use thereof
JP2022504663A (en) A plasmid containing a sequence encoding an mRNA with a segmented poly (A) tail
Madsen et al. The PARS sequence increase the efficiency of stable Pichia pastoris transformation
Putti et al. Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells
CN115011635A (en) Construction and application of dual-luciferase report system for siRNA rapid screening
JPH11332560A (en) Recombinant adenovirus, cosmid vector containing the recombinant virus genom and animal cell infected with the recombinant virus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22771004

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023506894

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22771004

Country of ref document: EP

Kind code of ref document: A1