Method for producing cultured avian cell lines, cultured cell lines for bioreactor evaluation, and bioreactor evaluation kit.

Abstract

To provide a bird cell line, a method for producing a cell line for bioreactor evaluation and a kit for bioreactor evaluation which make it possible to evaluate a bioreactor in a short time.SOLUTION: A bird cell line includes, in its genome, an exogenous promoter sequence for constant expression of at least one gene included in an in ovo protein gene locus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing cultured avian cell lines, cultured cell lines for evaluating bioreactors, and a kit for evaluating bioreactors. [Background technology]

[0002] Bird eggs are known to have a high protein content. For example, laying hens have high productivity, laying approximately 300 eggs per year. For this reason, bird eggs have been sought after as bioreactors for producing large quantities of useful proteins in egg whites. Seberipase alfa (Kanuma®), a drug used to treat congenital lysosomal acid lipase deficiency, is produced from chicken eggs. The chicken eggs used to produce seberipase alfa were created through genetic modification using a viral vector.

[0003] Patent documents 1 and 2 disclose that human interferon-beta can be expressed in chicken eggs by knocking in the human interferon-beta gene at the ovalbumin gene locus using genome editing technology.

[0004] To utilize bird eggs as a bioreactor, as in the production of seberipase alpha described above, and furthermore, the production of interferon β disclosed in Patent Documents 1 and 2, it is necessary to perform genetic recombination on early embryos or cultured primordial germ cells, or to knock in the genes of useful proteins into the egg protein locus using genome editing technology. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2015 / 199225 [Patent Document 2] International Publication No. 2017 / 111144 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The extent to which the knock-in gene product is contained in the egg, and whether the useful protein accumulated in the egg can maintain the desired activity, can only be confirmed after the bioreactor is actually established. Establishing a bioreactor requires steps such as culturing primordial germ cells, genetic modification, transplantation experiments, creation of germ cell chimeras, sexual maturation of germ cell chimeras, creation of G1 through mating, sexual maturation of G1, and creation of G2 through mating of G1. These steps take 2-3 years in chickens and over a year in quail. If there are problems with the quantity and activity of useful proteins after the bioreactor is established, the bioreactor construction must be restarted from the beginning.

[0007] Intraocular proteins such as ovalbumin and ovomucoid are produced by endocrine cells in the fallopian tubes in vivo. If the production of intraocular proteins could be studied in vitro, it would be possible to predict the quantity and activity of useful proteins that can be obtained from a bioreactor before commencing bioreactor construction. However, culturing endocrine cells that produce intraocular proteins is extremely difficult and has not been put into practical use.

[0008] This invention has been made in view of the above circumstances, and aims to provide a avian cell line that can evaluate a bioreactor in a short period of time, a method for producing a cell line for evaluating a bioreactor, and a kit for evaluating a bioreactor. [Means for solving the problem]

[0009] A cultured avian cell line according to the first aspect of the present invention is Ovalbumin To constitutively express at least one gene contained within a gene locus. First The genome contains foreign promoter sequences death, The aforementioned gene is, Contains the ovalbumin gene The first foreign promoter sequence is, Located upstream of exon 1 of the ovalbumin gene .

[0011] before said gene is 、 before at said ovalbumin locus Upstream or downstream of the open reading frame of the ovalbumin gene a knocked-in exogenous gene Includes , may be provided.

[0014] the First exogenous promoter sequence is an EF1α promoter sequence, may be provided.

[0015] O ovomucoid locus The genome further has a second exogenous promoter sequence that causes constitutive expression of at least one gene contained therein, before said gene constitutively expressed by the first exogenous promoter sequence is 、 before at said ovalbumin locus Upstream or downstream of the open reading frame of the ovalbumin gene a first knocked-in exogenous gene Further including , said gene constitutively expressed by the second exogenous promoter sequence is a second exogenous gene knocked-in upstream of the ovomucoid gene, That , The second exogenous promoter sequence is, Located upstream of the second foreign gene, may be provided.

[0016] The avian cultured cell line according to the first aspect of the present invention is a genetically modified DF-1 cell, may be provided.

[0017] A method for producing a cultured cell line for evaluating a bioreactor according to the second aspect of the present invention comprises: in the genome of an avian cultured cell line, Ovalbumin an exogenous promoter sequence that constitutively expresses at least one gene contained in a locus Upstream of exon 1 of the ovalbumin gene The knock-in step, The aforementioned Ovalbumin at the gene locus Upstream or downstream of the open reading frame of the ovalbumin gene The steps involve knocking in an exogenous gene, Includes fruit, The aforementioned gene is, Includes the ovalbumin gene and the aforementioned foreign gene nothing.

[0018] A bioreactor evaluation kit according to a third aspect of the present invention is: A cultured avian cell line according to the first aspect of the present invention described above, The aforementioned Ovalbumin at the gene locus Upstream or downstream of the open reading frame of the ovalbumin gene The aforementioned gene is a reagent for knocking in an exogenous gene, It is equipped with. [Effects of the Invention]

[0019] According to the present invention, bioreactors can be evaluated in a short period of time. [Brief explanation of the drawing]

[0020] [Figure 1] Figure A shows the distribution of exons at the ovalbumin locus in the genome of a wild-type cultured chicken cell line. Figure B illustrates the structure of the ovalbumin locus in the genome of a cultured cell line according to an embodiment of the present invention. Figure C shows the distribution of exon 1 at the ovomucoid locus in the genome of a wild-type cultured chicken cell line. Figure D illustrates the structure of the ovomucoid locus in the genome of a cultured cell line according to an embodiment of the present invention. [Figure 2]Figure A shows the target sequence of the guide RNA (gRNA) and the structure of the Precise integration into target chromosome (PITCh) vector for knocking in the EF1α promoter when the EF1α promoter is knocked in upstream of exon 1 of the ovalbumin locus according to Example 1. Base sequences written in uppercase indicate the exon base sequence, and base sequences written in lowercase indicate the intron base sequence. Figure B shows the structure of the ovalbumin locus with the EF1α promoter knocked in upstream of exon 1. Figure C shows the target sequence of the gRNA and the structure of the PITCh vector for knocking in the EF1α promoter when the EF1α promoter is knocked in between exon 1 and exon 2 of the ovalbumin locus according to Example 1. Base sequences written in uppercase indicate the exon base sequence, and base sequences written in lowercase indicate the intron base sequence. Figure D shows the structure of the ovalbumin locus with the EF1α promoter knocked in between exon 1 and exon 2. [Figure 3] Figures 2A and 2C show the vector maps used to construct the PITCh vectors. [Figure 4] Figure A shows the target sequence in the PITCh vector shown in Figure 2A. Figure B shows the target sequence in the PITCh vector shown in Figure 2B. [Figure 5] This figure shows the results of a polymerase chain reaction (PCR) to confirm that the EF1α promoter was knocked into the target region of the ovalbumin gene locus in Example 1. [Figure 6] This figure shows the PCR results to confirm that the EF1α promoter was knocked into the ovalbumin gene locus in Example 1. [Figure 7] This figure shows the results of reverse transcription PCR (RT-PCR) to confirm that the ovalbumin gene was transcribed in the cell line in which the EF1α promoter was knocked into the ovalbumin gene locus in Example 1. [Figure 8] This figure shows the results of quantitative RT-PCR (qRT-PCR) quantification of ovalbumin gene mRNA expression in cell lines in which the EF1α promoter was knocked into the ovalbumin gene locus in Example 1. [Figure 9] This figure shows the results of a Western blot to confirm the expression of ovalbumin protein in a cell line in which the EF1α promoter was knocked into the ovalbumin gene locus in Example 1. [Figure 10] This figure shows the concentration of ovalbumin protein measured by sandwich ELISA in a cell line in which the EF1α promoter was knocked into the ovalbumin gene locus in Example 1. [Figure 11] Figure A shows the location where the human fibroblast growth factor-2 (hFGF2) gene is knocked into the ovalbumin locus in which the EF1α promoter according to Example 1 is knocked in. Figure B shows the structure of the ovalbumin locus in which the hFGF2 gene is knocked in. Figure C shows the predicted mRNA sequence produced after transcription of the ovalbumin locus in which the hFGF2 gene is knocked in. [Figure 12] This figure shows a map of the vectors used to prepare the PITCh vector containing the hFGF2 gene according to Example 1. [Figure 13] This figure shows a map of the PITCh vector carrying the hFGF2 gene according to Example 1. [Figure 14] This figure shows a map of the vectors used to create the CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeat and Crisper associated protein) vector that cleaves the PITCh vector shown in Figure 13. [Figure 15] This figure shows the PCR results to confirm that hFGF2 according to Example 1 was knocked in downstream of the EF1α promoter. [Figure 16] This figure shows the results of RT-PCR to confirm that the ovalbumin gene and the hFGF2 gene were transcribed in Example 1. [Figure 17] This figure shows the concentration of ovalbumin protein measured after single cloning of a strain in which the hFGF2 gene was knocked into the ovalbumin gene locus in Example 1. [Figure 18] This figure shows the concentration of hFGF2 protein measured after single cloning of a strain in which the hFGF2 gene was knocked into the ovalbumin locus in Example 1. [Figure 19] Figure A shows a schematic of the EF1α promoter knock-in upstream of the ovomucoid gene according to Example 2, and the configuration of the PITCh vector for knocking in the EF1α promoter. Figure B shows the ovomucoid gene locus after knocking in the EF1α promoter. [Figure 20] Figures A and B show the PCR results for confirming the 5' and 3' junctions of the cassette containing the EF1α promoter knocked into the ovomucoid locus in Example 2, respectively. Figure C shows the genotyping results for the ovomucoid locus with the EF1α promoter knocked in. [Figure 21] This figure shows the results of RT-PCR to confirm that the ovomucoid gene was transcribed in the cell line in which the EF1α promoter was knocked into the ovomucoid gene locus in Example 2. [Figure 22] This figure shows the results of a Western blot to confirm the expression of ovomucoid protein in a cell line in which the EF1α promoter was knocked into the ovomucoid gene locus in Example 2. [Figure 23] This figure shows the concentration of ovomucoid protein measured by sandwich ELISA in cell lines in which the EF1α promoter was knocked into the ovomucoid gene locus in Example 2. [Figure 24]This figure shows an overview of the knock-in of a cassette containing the hFGF2 gene into an ovomucoid locus with the EF1α promoter knocked in according to Example 2, the target sequence of the gRNA, and the configuration of the PITCh vector for knocking in the hFGF2 gene. [Figure 25] This figure shows the ovomucoid locus in which the hFGF2 gene according to Example 2 was knocked in. [Figure 26] Figures A and B show the PCR results for confirming the 5' and 3' junctions of the cassette containing the hFGF2 gene knocked into the ovomucoid locus in Example 2, respectively. Figure C shows the genotyping results for the ovomucoid locus into which the hFGF2 gene was knocked. [Figure 27] This figure shows the results of a Western blot to confirm that ovalbumin protein is not expressed in a cell line in which the EF1α promoter and the hFGF2 gene were knocked into the ovalbumin gene locus according to Example 2. [Figure 28] This figure shows the concentration of ovalbumin protein measured by sandwich ELISA in a cell line in which the EF1α promoter and the hFGF2 gene were knocked into the ovalbumin gene locus according to Example 2. [Figure 29] This figure shows the target sequence of the genome editing tool when the hFGF2 gene is knocked in downstream of exon 8 of the ovalbumin gene locus according to Example 3. [Figure 30] This figure shows a map of the vectors used to prepare the donor vector according to Example 3. [Figure 31] This figure shows a map of the donor vectors according to Example 3. [Figure 32] Figure A shows the location where the hFGF2 gene is knocked into the ovalbumin locus by genome editing according to Example 3. Figure B shows the structure of the ovalbumin locus after the hFGF2 gene has been knocked in. Figure C shows the predicted mRNA sequence produced after transcription of the ovalbumin locus after the hFGF2 gene has been knocked in. [Figure 33] This figure shows the PCR results for confirming the hFGF2 gene knocked into the ovalbumin locus in Example 3. [Figure 34] This figure shows the PCR results to confirm that the hFGF2 gene was knocked in downstream of the EF1α promoter in Example 3. [Figure 35] This figure shows the results of RT-PCR to confirm that the ovalbumin gene and the hFGF2 gene were transcribed in Example 3. [Figure 36] This figure shows the concentration of ovalbumin protein measured after single cloning of a strain in which the hFGF2 gene was knocked into the ovalbumin gene locus in Example 3. [Figure 37] This figure shows the concentration of hFGF2 protein measured after single cloning of a strain in which the hFGF2 gene was knocked into the ovalbumin locus in Example 3. [Modes for carrying out the invention]

[0021] Embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments and drawings described below.

[0022] The avian cell line according to this embodiment is suitable for use in evaluation systems when using avian eggs as bioreactors for the production of proteins or peptides. Using this cell line, it is possible to confirm the amount of protein or peptide produced from the knocked-in foreign gene in the egg and whether the produced protein or peptide has the desired activity.

[0023] The avian cell line according to this embodiment has an exogenous promoter sequence in its genome that constitutively expresses at least one gene contained in an intraocular protein locus. The birds are not particularly limited and include, for example, chickens, ducks, turkeys, waterfowl, geese, quail, pheasants, parrots, finches, hawks, ostriches, emus, and cassowaries. Preferably, the bird is a chicken. The breed of chicken is not particularly limited and includes, for example, White Leghorn, Brown Leghorn, Barred Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partidge colored, and Korean Oge.

[0024] Any avian cell line can be used as a cultured cell line derived from birds, but a avian cell line used as a bioreactor is preferred. Examples of known cell lines that can be used include chicken embryo fibroblast cell lines and embryonic kidney cell lines. Preferably, the avian cell line is a genetically modified chicken embryo fibroblast cell line (DF-1 cells).

[0025] The intraocular protein gene can be any gene that codes for a protein expressed in a bird egg, such as genes that code for ovalbumin, ovomucoid, ovoinhibitor, ovoglobulin, lysozyme, ovomucin, and ovotransferrin. Preferably, the intraocular protein gene locus is at least one of the ovalbumin locus and the ovomucoid locus.

[0026] An oocyte protein locus is a genomic location upstream of the 5' untranslated region (5'UTR) that includes transcriptional regulatory regions such as promoters and enhancers involved in the expression of oocyte protein genes, the 5'UTR, the open reading frame (ORF) from the start codon to the stop codon, and the 3' untranslated region (3'UTR). For example, an oocyte protein locus is the region from the enhancer to the terminator that regulates the expression of oocyte protein genes. Note that "upstream" in the genome refers to the direction from the 5' end of the non-template strand (sense strand) of the genome, and the direction from upstream to downstream refers to the direction from 5' to 3' of the non-template strand of the genome.

[0027] An exogenous promoter sequence is a promoter sequence that does not originate from a cultured cell line. The expression of the above-mentioned oocyte protein gene is controlled by the promoter, and the oocyte protein is expressed specifically in the fallopian tube. In contrast, an exogenous promoter sequence causes constitutive (or forced) expression of the gene contained in the oocyte protein gene locus. Constitutive expression means that the gene is always expressed, rather than being limited expression such as tissue-specific expression. A promoter sequence that causes constitutive (ubiquitous) expression of a gene is preferably a known promoter that functions in mammalian cells, such as CMV, EF1α, EFS, CAG, CBh, SFFV, MSCV, SV40, mPGK, hPGK, and UBC. Preferably, the exogenous promoter sequence is the EF1α promoter sequence.

[0028] The foreign promoter sequence can be knocked in at any position of the gene, as long as the gene contained in the ovalbumin protein locus is constitutively expressed. For example, if the ovalbumin protein locus is the ovalbumin gene locus, the foreign promoter sequence is preferably located upstream of exon 1 of the ovalbumin gene. Figure 1A shows the ovalbumin gene locus in the genome of a wild-type cultured avian cell line. In contrast, the cultured cell line according to this embodiment has the foreign promoter sequence upstream of exon 1 of the ovalbumin gene locus in the genome, as shown in Figure 1B. Since the start codon of the ovalbumin gene is in exon 2, if the ovalbumin protein locus is the ovalbumin gene locus, the foreign gene may be located upstream of exon 2 of the ovalbumin gene, that is, between exon 1 and exon 2, upstream of the start codon of the ovalbumin gene.

[0029] The avian cell line according to this embodiment may have a marker gene at the intraocular protein locus. When knocking in an exogenous promoter sequence by genome editing, etc., it is preferable to stably incorporate marker gene 1 into the genome together with the exogenous promoter sequence, as shown in Figure 1B, in order to select the knocked-in cell line. Marker gene 1 is a gene for a fluorescent protein, a gene for an enzyme that catalyzes a color reaction, and a drug resistance gene, etc. Examples of fluorescent proteins and enzymes that catalyze color reactions include green fluorescent protein, luciferase, β-glucuronidase, and β-galactosidase. An example of a drug resistance gene is the neomycin resistance gene (Neo R ), Hygromycin resistance gene (Hyg R ), puromycin resistance gene (Puro R ), blastosis gene (Blast R ) and zeosin resistance gene (Zeo R Examples include:

[0030] When the intraocular protein locus is the ovalbumin locus, the genes included in the intraocular protein locus that are constitutively expressed by the foreign promoter sequence are preferably the ovalbumin gene and the knocked-in foreign gene. The foreign gene is a gene that codes for any useful protein or useful peptide (hereinafter also collectively referred to as "useful proteins, etc.") produced in a bioreactor via avian eggs. The useful proteins, etc. coded by the foreign gene are preferably secreted proteins or peptides. Examples of useful proteins, etc. include antibodies, scFv, Fab, Fab', F(ab')2, Fv, single-chain antibodies, fragments of antibodies such as scFv and dsFv, enzymes, hormones, growth factors, cytokines, interferons, collagen, extracellular matrix molecules, vaccine antigens, agonist proteins and antagonist proteins, etc. For example, a useful protein is hFGF2. When the protein coded by the foreign gene is a physiologically active protein used as a pharmaceutical drug administered to humans, the foreign gene is of mammalian origin, preferably human origin. Furthermore, in the case of industrially usable proteins such as protein A and the proteins that make up spider silk, the foreign gene may encode a protein derived from any organism, including microorganisms such as bacteria and yeast, plants and animals, or an artificial protein.

[0031] The foreign gene can be knocked in at any position on the oocyte protein locus, as long as it is constitutively expressed by the foreign promoter sequence. For example, the foreign gene can be knocked in upstream or downstream of the ORF of the oocyte protein gene. Preferably, as shown in Figure 1B, the foreign gene is knocked in upstream of the ovalbumin gene. Since ovalbumin is an essential protein in the fallopian tube, if the oocyte protein locus is the ovalbumin locus, it is preferable that the ovalbumin gene is expressed in addition to the foreign gene. Marker gene 2 may be incorporated along with the foreign gene to select cell lines in which the foreign gene has been knocked in.

[0032] When a foreign gene is knocked in upstream of an intrinsic ovarian protein gene at an ovarian protein locus, a linker may be inserted between the foreign gene and the ovarian protein gene to control the expression of multiple open reading frames (ORFs) with a single promoter. The linker may be, for example, an internal ribosome entry site (IRES) corresponding to an RNA element that enables cap-independent translation initiation, or a nucleotide sequence corresponding to a 2A peptide that induces ribosome skipping during intracellular protein translation. As shown in Figure 1B, by inserting an IRES, the foreign gene and the ovalbumin gene are expressed under the control of the foreign promoter sequence. When a 2A peptide is used, a protein fused with the foreign gene and the ovarian protein gene is expressed, and it is cleaved at the 2A peptide portion to produce the protein encoded by the foreign gene and the ovarian protein. Examples of 2A peptides include T2A, P2A, E2A, and F2A.

[0033] The ovomucoid gene locus may also be the ovomucoid gene locus. Figure 1C shows a portion of the ovomucoid gene locus in the genome of a wild-type cultured avian cell line. When the ovomucoid gene locus is the ovomucoid gene locus, as shown in Figure 1C, the gene included in the ovomucoid gene locus that is constitutively expressed by the foreign promoter sequence in the cultured cell line according to this embodiment is a foreign gene knocked in upstream of the ovomucoid gene. In this case, if the above linker is not inserted between the foreign gene and the ovomucoid gene, only the foreign gene will be transcribed, the ovomucoid gene will not be transcribed, and the ovomucoid gene can be knocked off. Since ovomucoid is not essential in the oviduct and can also be an allergen, it is not necessary for ovomucoid to be produced. The foreign gene may be knocked in at the position of the start codon in exon 1 of the ovomucoid gene.

[0034] The oocyte protein locus in this embodiment is not limited to one; genes encoding useful proteins, etc., may be knocked into each of the oocyte protein locus 3 and the oocyte protein locus 4, which is different from oocyte protein locus 3. For example, oocyte protein locus 3 is the ovalbumin locus, and oocyte protein locus 4 is the ovomucoid locus. In the following, the case in which oocyte protein locus 3 and oocyte protein locus 4 are the ovalbumin locus and the ovomucoid locus, respectively, will be explained as an example.

[0035] When using the ovalbumin locus and the ovomucoid locus, the foreign promoter sequences are foreign promoter sequence 5 (first foreign promoter sequence) which constitutively expresses at least one gene contained in the ovalbumin locus, and foreign promoter sequence 6 (second foreign promoter sequence) which constitutively expresses at least one gene contained in the ovomucoid locus. Foreign promoter sequence 5 and foreign promoter sequence 6 may be the same type of promoter sequence or different types of promoter sequences. For example, both foreign promoter sequence 5 and foreign promoter sequence 6 are EF1α promoter sequences.

[0036] The genes constitutively expressed by foreign promoter sequence 5 are the ovalbumin gene and foreign gene 7 (the first foreign gene) knocked into the ovalbumin locus. The gene constitutively expressed by foreign promoter sequence 6 is foreign gene 8 (the second foreign gene) knocked into the upstream of the ovomucoid gene.

[0037] By using foreign gene 7 and foreign gene 8 to encode the same useful protein, the expression level of the useful protein can be increased compared to using a single intraocular protein locus. Furthermore, by using foreign gene 7 and foreign gene 8 to encode different useful proteins, multiple types of useful proteins can be expressed.

[0038] Next, a method for producing a cultured avian cell line according to this embodiment will be described. The method for producing the cultured cell line includes a first step of knocking in an exogenous promoter sequence that causes constitutive expression of at least one gene contained in an egg protein locus into the genome of a cultured avian cell line, and a second step of knocking in an exogenous gene into an egg protein locus.

[0039] The avian cell line targeted for knock-in is preferably a wild-type cell line that has not undergone artificial genetic modification. Preferably, the avian cell line targeted for knock-in is DF-1 cells. By using DF-1 cells in the method for producing the avian cell line according to this embodiment, genetically modified DF-1 cells can be obtained.

[0040] Knock-in of foreign promoter sequences and foreign genes into the genome can be performed using known genome editing technologies. Genome editing is a technology that modifies genes by utilizing the break and repair of double-stranded DNA. For example, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and the CRISPR / Cas system can be used for genome editing.

[0041] ZFNs and TALENs are polypeptides consisting of a DNA-binding domain and a DNA-cleaving domain. TALENs and ZFNs cleave double-stranded DNA by forming a dimer at the binding site of the DNA-binding domain, where a pair of DNA-cleaving domains meet in close proximity. The DNA-binding domain contains multiple repeating DNA-binding modules, each recognizing a specific base pair of DNA. By appropriately designing the DNA-binding modules, specific cleavage of target base sequences in the genome is possible. In ZFNs, the DNA-binding domain and DNA-cleaving domain are the zinc finger motif and FokI, respectively. In TALENs, the DNA-binding domain and DNA-cleaving domain are the diFokI and TAL effector, respectively.

[0042] CRISPR / Cas9, which uses Cas9 as a Cas protein with nuclease activity, is a gene modification tool that can specifically cleave double-stranded DNA between protospacer adjacent motifs (PAM sequences) and adjacent bases at the 5' end of the genome, thereby modifying any part of the genome.

[0043] The CRISPR / Cas9 system uses the Cas9 protein and gRNA. The gRNA contains a base sequence that forms a base pair with the complementary strand of the target sequence, corresponding to CRISPR RNA (crRNA), and a base sequence that functions as a transactivating crRNA (tracrRNA), serving as a scaffold for Cas9 protein binding. The Cas9-gRNA complex is formed when the gRNA base pairs with the complementary strand of the target sequence. The Cas9 protein then cleaves the double-stranded DNA.

[0044] Breaks in double-stranded DNA can lead to the loss of a large amount of genetic information or cancer, so they are repaired very rapidly within cells. Preferably, in the method for producing cultured cell lines according to this embodiment, the knock-in of foreign promoter sequences and foreign genes is performed via the PITCh system. The PITCh system inserts foreign promoter sequences and foreign genes using microhomology-mediated end-joining (MMEJ), one of the repair mechanisms for broken double-stranded DNA. MMEJ is a mechanism in which complementary sequences (5-25 base pairs) are joined between the two ends of a double-strand break, and repair occurs.

[0045] When knocking in a foreign promoter sequence into the genome using the CRISPR / Cas9 system and the PITCh system, a vector expressing CRISPR / Cas9 that cleaves double-stranded DNA at the oocyte protein locus, a donor vector containing the foreign promoter sequence, and a vector expressing CRISPR / Cas9 that cleaves the donor vector should be introduced into a cultured cell line. These vectors can be introduced into the cultured cell line by known methods such as microinjection, electroporation, calcium phosphate, and lipofection. Similarly, when knocking in a foreign gene into the genome, a vector expressing CRISPR / Cas9 that cleaves double-stranded DNA at the oocyte protein locus, a donor vector containing the foreign gene, and a vector expressing CRISPR / Cas9 that cleaves the donor vector should be introduced into the cell line in which the foreign promoter sequence has been introduced into the genome in the first step.

[0046] In the first and second steps, marker gene 1 and marker gene 2 may be knocked in along with the foreign promoter sequence and foreign gene, respectively. This allows for the selection of cultured cell lines (clones) in which the foreign promoter sequence and foreign gene have been knocked into the genome, via the marker genes introduced into the genome along with the foreign promoter sequence and foreign gene. It is preferable that marker gene 2, which is knocked in along with the foreign gene, is different from marker gene 1, which is knocked in along with the foreign promoter sequence.

[0047] The first and second steps may be performed simultaneously. That is, the vector used for knocking in an exogenous promoter sequence and the vector used for knocking in an exogenous gene may be introduced into a cultured cell line.

[0048] To confirm that the knocked-in foreign promoter sequence constitutively expresses at least one gene contained within the oocyte protein gene locus, the expression of mRNA transcribed from the oocyte protein gene or the oocyte protein itself can be evaluated in the cultured cell line obtained in the first step using known methods. mRNA expression can be confirmed by extracting RNA from the cultured cell line and using known methods such as PCR, real-time PCR, quantitative PCR, and sequence analysis of the transcript. Oocyte protein expression can be confirmed in samples prepared from the culture supernatant or lysate of the cultured cell line using known methods such as Western blotting and ELISA. The expression of the knocked-in foreign gene can be confirmed in the cultured cell line obtained in the second step in the same way as the expression of the oocyte protein gene described above.

[0049] When knocking in foreign genes to multiple oocyte protein loci, in the first step described above, a foreign promoter sequence 5 is knocked into the genome of a cultured avian cell line to constitutively express at least one gene contained in oocyte protein locus 3. Then, in the second step described above, foreign gene 7 is knocked into oocyte protein locus 3. Furthermore, the first and second steps can be repeated for oocyte protein locus 4. Specifically, as the third step, a foreign promoter sequence 6 is knocked into the genome of the cultured cell line in which foreign gene 7 has been knocked in, to constitutively express at least one gene contained in oocyte protein locus 4. Subsequently, as the fourth step, foreign gene 8 can be knocked into oocyte protein locus 4.

[0050] According to the cultured cell line of this embodiment, ovalbumin and ovomucoid, which are expressed in tubal endocrine cells that are not currently in practical use, can be expressed in cell lines other than tubal endocrine cells. Therefore, it is possible to examine the expression level of useful proteins encoded by foreign genes knocked into the oval protein gene locus, and whether the useful proteins maintain the desired activity. If the desired expression and activity of useful proteins are confirmed in the genetically modified cultured cell line, it is thought that the foreign gene knocked into the same position at the oval protein gene locus will be similarly expressed in tubal endocrine cells. Therefore, the production amount and activity of useful proteins by the bioreactor can be evaluated in a short period of time before commencing the construction of the bioreactor.

[0051] When constructing a bioreactor based on evaluation using the cultured cell line according to this embodiment, the foreign gene should be knocked in as described above at the location of the intraocular protein gene locus determined in the study of the cultured cell line, using avian embryonic stem cells, particularly pluripotent stem cells derived from the upper blast layer, or primordial germ cells. Subsequently, the primordial germ cells with the knocked-in foreign gene are transplanted into an avian embryo. The transplantation procedure is not particularly limited, but the primordial germ cells can be injected into the avian embryo using a tubule. After transplanting the primordial germ cells, a chimeric individual can be produced by hatching the egg containing the transplanted embryo. By mating the resulting chimeric individual, genetically modified birds that inherit a genome homozygous for the foreign gene knocked in at the intraocular protein gene locus can be produced with a high probability.

[0052] As described above, the method for producing cultured cell lines according to this embodiment can be incorporated into a method for constructing a bioreactor. Therefore, in another embodiment, a method for constructing a bioreactor is provided, comprising the first step and the second step described above.

[0053] In another embodiment, a bioreactor evaluation kit is provided. This kit comprises a cultured avian cell line having an exogenous promoter sequence in its genome that constitutively expresses at least one gene located in an egg protein locus, and a reagent for knocking in the exogenous gene as the gene at the egg protein locus. The genome of the cultured cell line included in the kit does not have the exogenous gene knocked in, only the exogenous promoter sequence. The reagent for knocking in the exogenous gene is, for example, the reagent used in the second step above for knocking in the exogenous gene by genome editing technology. More specifically, the reagent may include a vector expressing CRISPR / Cas9 that cleaves double-stranded DNA at the egg protein locus, a donor vector (PITCh vector) into which the exogenous gene can be incorporated, and a vector expressing CRISPR / Cas9 that cleaves the donor vector.

[0054] The bioreactor evaluation kit may further include a buffer, a drug for selection using a marker gene, and various primers for confirming knock-in. [Examples]

[0055] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0056] [Example 1: Genome editing of the ovalbumin (OVA) gene locus] 1. Development of genome editing tools To knock in a forced expression promoter (foreign promoter sequence) into the OVA locus using the CRISPR / Cas9 system, we created a genome editing tool. The PITCh system, a highly efficient knock-in technology, was used as the knock-in method (Nakade et al., Nat Commun, 2014). The genome editing tool used in the PITCh system consists of three types: a CRISPR / Cas9 vector targeting the OVA locus, a donor vector (PITCh vector) containing the EF1α promoter as the foreign promoter sequence, and a CRISPR / Cas9 vector for cleaving the PITCh vector.

[0057] (Construction of CRISPR / Cas9 vectors targeting the OVA gene locus) Figure 2A shows the PAM sequence and gRNA target sequence (underlined) when targeting the upstream region of exon 1 of the OVA gene (hereinafter also referred to as "exon 1 editing"). On the other hand, Figure 2C shows the PAM sequence and gRNA target sequence (underlined) when targeting the region between exon 1 and exon 2 (hereinafter also referred to as "exon 2 editing"). Annealed synthetic oligonucleotides, which serve as templates for gRNA, were synthesized (Eurofins Genomics). The nucleotide sequences of the synthetic oligonucleotides for exon 1 editing are shown in SEQ ID NOs. 3 and 4. The nucleotide sequences of the synthetic oligonucleotides for exon 2 editing are shown in SEQ ID NOs. 5 and 6.

[0058] Annealed synthetic oligonucleotides were inserted into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene, #42230, hereinafter also referred to as the "pX330 vector") using the Golden Gate method with BpiI (Thermo Fisher Scientific) and Ligation high Ver.2 (TOYOBO). The reaction mixture was transformed into Competent Quick DH5 (TOYOBO), and after small culture, the plasmid was extracted (FastGene Plasmid MiniKit, Nippon Genetics). Subsequently, the extracted plasmid was used as a template for sequencing analysis (BigDye® Terminator v3.1 Cycle Sequencing Kit, Thermo Fisher Scientific). Sequencing analysis confirmed that the synthetic oligonucleotides were inserted into the pX330 vector.

[0059] (Preparation of PITCh vectors equipped with the EF1α promoter) Figure 3 shows a vector map of the pBApo-EF1α Pur vector (manufactured by Takara Bio Inc.) used to construct the PITCh vector. The PITCh vector in this example was constructed by inserting a nucleotide sequence of approximately 20 bp homologous to the OVA gene (microhomology sequence) between BamHI and HindIII in the cloning site of the pBApo-EF1α Pur vector. The nucleotide sequence to be inserted was synthesized as a synthetic oligonucleotide, and the synthetic oligonucleotide was annealed in the same manner as when constructing the CRISPR / Cas9 vector. The nucleotide sequences of the synthetic oligonucleotide in exon 1 editing are shown in SEQ ID NOs. 7 and 8. The nucleotide sequences of the synthetic oligonucleotide in exon 2 editing are shown in SEQ ID NOs. 9 and 10. The nucleotide sequence of the synthetic oligonucleotide used in exon 1 editing includes restriction enzyme sites (BamHI site and HindIII site) and a microhomology sequence. Since exon 2 contains a start codon, the nucleotide sequence of the synthetic oligonucleotide in exon 2 editing includes a Kozak sequence in addition to the restriction enzyme site and microhomology sequence.

[0060] The pBApo-EF1α Pur vector was restricted enzyme-treated with BamHI and HindIII, and then electrophoresed on a 1% agarose gel. The target vector was then excised and purified (FastGene Gel / PCR Extraction Kit, Nippon Genetics Co., Ltd.). The purified vector and insert were ligated using Ligation High Ver.2. These were then transformed into competent cells as described above and subjected to sequencing analysis. Sequencing analysis confirmed that the synthetic oligonucleotide was inserted into the cloning site of the pBApo-EF1α Pur vector.

[0061] (Preparation of CRISPR / Cas9 vectors to cleave PITCh vectors) The target sequences in the PITCh vectors prepared for exon 1 editing and exon 2 editing are shown in Figures 4A and 4B, respectively. Similar to the preparation of the CRISPR / Cas9 vector targeting the OVA locus described above, annealed synthetic oligonucleotides serving as gRNA templates were synthesized. The nucleotide sequences of the synthetic oligonucleotides for exon 1 editing are shown in SEQ ID NOs. 11 and 12. The nucleotide sequences of the synthetic oligonucleotides for exon 2 editing are shown in SEQ ID NOs. 13 and 14. After annealing, the synthetic oligonucleotides were inserted into the pX330 vector, and their insertion was confirmed by sequencing analysis.

[0062] 2. Creation of OVA-expressing cell lines using genome editing technology As described above, a DNA solution containing a CRISPR / Cas9 vector targeting the OVA locus, a PITCh vector equipped with an EF1α promoter, and a CRISPR / Cas9 vector that cleaves the PITCh vector was introduced into DF-1 cells (ATCC® CRL-12203®). DF-1 cells were maintained using 10% fetal bovine serum (FBS) (Biological Industries)-KnockOut® DMEM (Dulbecco's modified Eagle medium) (Thermo Fisher Scientific) and 1% GlutaMax® (Thermo Fisher Scientific). Lipofectamine® 3000 Reagent (Thermo Fisher Scientific) was used for gene transfer according to the protocol. 24 hours after gene transfer, puromycin was added at a concentration of 1.0 μg / mL and the cells were cultured for 2 weeks.

[0063] To confirm EF1α promoter knock-in, genomic DNA was extracted from polyclonal cells (Puregene Cell Kit, QIAGEN) and subjected to genomic PCR (KOD One® PCR Master Mix, TOYOBO). Figure 2B shows the nucleotide sequences of forward primer P1 and reverse primer P2 related to exon 1 editing, respectively, as shown by SEQ ID NOs. 15 and 16. Figure 2D shows the nucleotide sequences of forward primer P3 and reverse primer P4 related to exon 2 editing, respectively, as shown by SEQ ID NOs. 17 and 18. As shown in Figure 5, the target bands were detected for both exon 1 and exon 2 editing. Subsequently, the cells were monocloned using the limiting dilution method and subjected to further analysis.

[0064] To determine whether knock-in occurred in one allele or both alleles, primers were designed on the OVA locus to sandwich the knock-in region. As shown in Figure 2B, the primers for exon 1 editing were forward primer P1 and reverse primer P5, whose nucleotide sequence is shown in SEQ ID NO: 19. As shown in Figure 2D, the nucleotide sequences of forward primer P6 and reverse primer P7 for exon 2 editing are shown in SEQ ID NOs: 20 and 21, respectively. Genomic PCR using these primers revealed high and low molecular weight bands for both exon 1 and exon 2 editing, as shown in Figure 6. This clearly indicates that the EF1α promoter was inserted into one allele as a result of the introduction of the genome editing tool.

[0065] 3. Confirmation of the presence or absence of off-target effects. We checked for off-target effects in strains in which the EF1α promoter established above was knocked in upstream of exon 1 of the OVA gene (hereinafter referred to as "EF1α-Exon1 strain") and strains in which the EF1α promoter was knocked in between exon 1 and exon 2 (hereinafter referred to as "EF1α-Exon2 strain"). First, we obtained information on chicken genome regions in which the combined 12 base pairs of the PAM and gRNA target sequences matched using CRISPR direct. Annotation was performed on these genome regions using the University of California SANTA CRUZ (UCSC) Genome Browser. Among the genome regions obtained by CRISPR direct, gene-coding regions were designated as candidate regions, and the presence or absence of off-target effects in these candidate regions was checked. The candidate regions are shown in Table 1.

[0066] [Table 1]

[0067] Primers were designed to amplify each candidate region. The nucleotide sequences of the forward and reverse primers for DACH1 are shown in SEQ ID NOs. 32 and 33, respectively. The nucleotide sequences of the forward and reverse primers for IFT140 are shown in SEQ ID NOs. 34 and 35, respectively. The nucleotide sequences of the forward and reverse primers for TRAIP are shown in SEQ ID NOs. 36 and 37, respectively. The nucleotide sequences of the forward and reverse primers for SNX2 are shown in SEQ ID NOs. 38 and 39, respectively. The nucleotide sequences of the forward and reverse primers for ATP2A2 are shown in SEQ ID NOs. 40 and 41, respectively. The nucleotide sequences of the forward and reverse primers for KIF4B are shown in SEQ ID NOs. 42 and 43, respectively.

[0068] Candidate regions were amplified from genomic DNA extracted from single-clonal cells of EF1α-Exon1 and EF1α-Exon2 strains, as well as from wild-type DF-1 cells used as a control. Direct sequencing was performed using these as templates, and sequence analysis was conducted. As a result, no mutations were found in a total of six off-target candidate regions.

[0069] 4. OVA expression analysis We investigated the expression of the OVA gene in the established EF1α-Exon1 and EF1α-Exon2 strains. First, to confirm whether or not the OVA gene was being transcribed, we performed RT-PCR, qRT-PCR, and sequencing analysis of the OVA gene transcript.

[0070] RNA was extracted from cultured EF1α-Exon1 and EF1α-Exon2 strains (FastGene™ RNA Premium Kit, Genetics Japan), and cDNA was synthesized (SuperScript IV reverse transcriptase, Thermo Fisher Scientific). RT-PCR was performed using the synthesized cDNA. The nucleotide sequences of the forward and reverse primers used are shown in SEQ ID NOs. 44 and 45, respectively. As shown in Figure 7, the RT-PCR results confirmed that the OVA gene was transcribed in each strain.

[0071] Next, the expression intensity of the OVA gene in EF1α-Exon1 and EF1α-Exon2 strains was evaluated by qRT-PCR. KOD SYBR qPCR mix (TOYOBO) was used for qRT-PCR. As a control, the expression level of β-actin was quantified. The nucleotide sequences of the forward and reverse primers for OVA are shown in SEQ ID NOs. 46 and 47, respectively. The nucleotide sequences of the forward and reverse primers for β-actin are shown in SEQ ID NOs. 48 and 49, respectively. As shown in Figure 8, the qRT-PCR results showed that the expression intensity in EF1α-Exon1 strain was approximately three times higher than that in EF1α-Exon2 strain.

[0072] To investigate whether normal transcription of the OVA gene was occurring in EF1α-Exon1 and EF1α-Exon2 cell lines, sequencing analysis of the OVA gene transcript was performed. First, the full length of the predicted OVA gene transcript was amplified by PCR using cDNA synthesized from each cell line as a template. Subsequently, direct sequencing was performed using the PCR product to analyze the OVA gene transcript. The base sequences of the primers used are shown in SEQ ID NOs. 50-55. The sequencing analysis confirmed normal transcription of the OVA gene in both EF1α-Exon1 and EF1α-Exon2 cell lines.

[0073] Western blotting and ELISA were performed to determine whether the OVA protein was expressed in EF1α-Exon1 and EF1α-Exon2 strains. For Western blotting, the culture supernatant and cell lysates of each strain were used as samples. The culture supernatant, cultured for two weeks, was concentrated approximately 2-fold using Amicon Ultra 30K (Merck) and used as the culture supernatant sample.

[0074] Lysate samples were prepared using the Nuclear Extract Kit (ACTIVE MOTIF) according to the protocol. Each sample was separated using a 15% polyacrylamide gel, transferred to a PVDF membrane, and blocked overnight at 4°C (PVDF Blocking Reagent for Can Get Signal®, TOYOBO). OVA proteins on the membrane were detected using anti-OVA rabbit antibodies (Egg Western Blotting Kit (egg albumin, Morinaga Biosciences Laboratories)). These were diluted 1:1000 with primary antibody Solution 1 (Can Get Signal® Immunoreaction Enhancer Solution, TOYOBO). HRP-labeled anti-rabbit / goat antibodies were used as secondary antibodies and diluted 1:5000 with secondary antibody Solution 2 (Can Get Signal® Immunoreaction Enhancer Solution, TOYOBO). Chemiluminescence was detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare). As shown in Figure 9, OVA proteins were detected in the culture supernatant sample of the EF1α-Exon1 strain.

[0075] For the ELISA method, culture supernatants and lysate samples were used from EF1α-Exon1 and EF1α-Exon2 strains cultured for 4 days. Lysate samples were prepared using the Nuclear Extract Kit. A sandwich ELISA method was employed, using two types of antibodies: a capture antibody and a detection antibody. Anti-OVALBUMIN (Hen Egg White) (RABBIT) Antibody (ROCKLAND) was used as the capture antibody. The capture antibody was diluted 1:2000 with PBS and immobilized overnight at 4°C. OVA standard samples were prepared by diluting the OVA standard samples from the ITEA Egg White Albumin (OVA) ELISA Kit (ITEA) with PBS at concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng / ml.

[0076] The detection antibody used was an anti-OVA rabbit antibody labeled with the Peroxidase Labeling Kit (Dojindo Molecular Technologies). The antibody used was an Egg Western Blotting Kit (egg albumin, Morinaga Institute of Biosciences) diluted 1:1000 with Solution 1 for primary antibodies. SureBlue Reserve™ TMB Microwell Peroxidase Substrate (SeraCare Life Sciences) was used as the substrate, and color development was detected using Multiskan Sky T (Thermo Fisher Scientific). Based on a calibration curve created from OVA standard samples, the OVA concentration was determined, and as shown in Figure 10, it was found that the culture supernatant sample of the EF1α-Exon1 strain contained a large amount of OVA protein, consistent with the results of the Western blot method.

[0077] 5. Introduction and expression analysis of useful protein genes Expression analysis of the OVA gene revealed that the OVA gene is highly expressed in the EF1α-Exon1 strain; therefore, the EF1α-Exon1 strain was used for introducing useful protein genes. As shown in Figures 11A and 11B, the hFGF2 gene, which encodes hFGF2, a useful protein used in regenerative medicine, was knocked in between exon 1 and exon 2 of the OVA locus in the EF1α-Exon1 strain. The PITCh system was also used for the knock-in of the hFGF2 gene into the OVA locus. The genome editing tools used in the PITCh system consist of three types: a CRISPR / Cas9 vector targeting the OVA locus, a donor vector containing the hFGF2 gene (PITCh vector), and a CRISPR / Cas9 vector for cleaving the PITCh vector. As the CRISPR / Cas9 vector targeting the OVA locus, the CRISPR / Cas9 vector targeting the OVA locus in the exon 2 editing described above was used.

[0078] (Construction of a PITCh vector containing the hFGF2 gene) For the preparation of the PITCh vector, we used pCRIS-PITChv2-FBL (Addgene, #63672) (Sakuma et al., Nat Protoc, 2016), as shown in Figure 12. The PITCh vector was constructed by rearranging the PITCh gRNA target sites of pCRIS-PITChv2-FBL with hFGF2-NeoR-IRES, as shown in Figure 13. The necessary genes were amplified by PCR using the specified primers, and after an in-fusion reaction (In-Fusion® HD Cloning Kit, Takara Bio Inc.), competent cells were transformed. After small culture, plasmids were extracted and sequenced to confirm the integration of the hFGF2-NeoR-IRES cassette. The nucleotide sequences of the primers used for PITCh vector preparation are shown in Table 2.

[0079] [Table 2]

[0080] (Preparation of CRISPR / Cas9 vectors to cleave PITCh vectors) The CRISPR / Cas9 vector used to cleave the PITCh vector was pX330S-2-PITCh (Addgene, #63670) (Sakuma et al., Nat Protoc, 2016), as shown in Figure 14.

[0081] The constructed genome editing tool was introduced into the EF1α-Exon1 cell line, and drug selection was performed with 100 μg / mL neomycin for two weeks. Genomic DNA was extracted from polyclonal cells and subjected to genomic PCR. The nucleotide sequences of forward primer P8 and reverse primer P9 for hFGF2 gene knock-in detection are shown in SEQ ID NOs. 64 and 65, respectively.

[0082] As shown in Figure 15, we confirmed that hFGF2 was knocked in downstream of the EF1α promoter. Subsequently, we monocloned the cells using the limiting dilution method and performed sequencing analysis to confirm that the hFGF2 gene was knocked in at the target position of the OVA locus.

[0083] Three strains (clones #30, #46, and #52) were obtained by monocloning, in which the hFGF2 gene was knocked into the target position of the OVA locus. Clones #30, #46, and #52 were cultured, RNA was extracted, and cDNA was synthesized. RT-PCR was performed using the synthesized cDNA. The predicted sequences after splicing in the three strains are shown in Figure 11C. The size of the augmented byproducts obtained by RT-PCR using forward primers (Fwd1) and reverse primers (Rev1), whose nucleotide sequences are shown in SEQ ID NOs. 66 and 67, respectively, was 2472 bp. As a result of RT-PCR, as shown in Figure 16, it was confirmed that the OVA gene and the hFGF2 gene were transcribed in clones #30, #46, and #52.

[0084] An ELISA method was performed to determine whether OVA protein and hFGF2 protein were expressed in clones #30, #46, and #52. For OVA protein detection, the culture supernatant from clones #30, #46, and #52 cultured for 7 days was used. For FGF2 protein detection, lysate samples from clones #30, #46, and #52 were used. Lysate samples were prepared according to the Human FGF2 sandwich ELISA kit (Proteintech). Figures 17 and 18 show the concentrations of OVA protein and hFGF2 protein, respectively. OVA protein was present in the culture supernatant at concentrations of 30 ng / ml to 50 ng / ml. hFGF2 protein was present in the lysate at concentrations of 1200 pg / ml to 1900 pg / ml.

[0085] [Example 2: Genome editing of the ovomucoid (OVM) gene locus] 1. Vector construction for generating OVM-expressing cells We established an OVM-expressing cell line by performing genome editing on DF-1 cells. Specifically, we stimulated the transcription and translation of the OVM gene by inserting the EF1α promoter using a CRISPR / Cas9-based PITCh system, targeting the upstream region of the OVM gene.

[0086] In establishing OVM-expressing cells, a PITCh vector and two types of CRISPR / Cas9 vectors were constructed. As shown in Figure 19A, the PITCh vector was loaded with the EF1α promoter, a marker gene, and the microhomology sequences required for the PITCh system. The two CRISPR / Cas9 vectors targeted the upstream of the OVM gene and the microhomology sequence of the PITCh vector, respectively. In Figure 19A, the underlined nucleotide sequences indicate the target sequences of the gRNA, and the arrows indicate the cleavage sites.

[0087] (Construction of CRISPR / Cas9 vectors) The pX330-U6-Chimeric BB-CBh-SpCas9 vector was used to construct the CRISPR / Cas9 vector. It was prepared by inserting an annealed synthetic oligonucleotide, which serves as a gRNA template, into the linearized pX330-U6-Chimeric BB-CBh-SpCas9 vector using BpiI. The method for annealing the synthetic oligonucleotide is described below.

[0088] The synthetic oligo was annealed by reacting an annealing solution containing 1 μL of 10× buffer (400 mM Tris-HCl (pH 8), 200 mM MgCl2, 500 mM NaCl), 1 μL of 100 μM sense oligo, 1 μL of 100 μM antisense oligo, and 7 μL of sterile distilled water at 95°C for 5 minutes, followed by cooling to 25°C over 90 minutes. The annealed synthetic oligo was then inserted into the pX330-U6-Chimeric BB-CBh-SpCas9 vector using the procedure described below. The composition of the reaction solution for annealing the oligo is shown below. pX330-U6-Chimeric BB-CBh-SpCas9(25ng / μL) 0.3μL Annealed synthetic oligonucleotide 0.5 μL Ligation high ver2 (manufactured by TOYOBO) 1.0 μL BpiI 0.1 μL 0.1 μL of sterile distilled water

[0089] Annealing oligonucleotides were inserted into the linearization vector by performing a reaction at 37°C for 5 minutes and at 16°C for 10 minutes for three cycles. The nucleotide sequences of the sense oligonucleotide and antisense oligonucleotide contained in the synthetic oligo targeting the upstream of the OVM gene are shown in SEQ ID NOs. 72 and 73, respectively. The nucleotide sequences of the sense oligonucleotide and antisense oligonucleotide contained in the synthetic oligo targeting the microhomology sequence of the PITCh vector are shown in SEQ ID NOs. 74 and 75, respectively. Hereafter, the CRISPR / Cas9 vector targeting the upstream of the OVM gene will be referred to as pX330-OVM 5'-UTR, and the CRISPR / Cas9 vector targeting the PITCh vector will be referred to as pX330-EF1α donor.

[0090] (Construction of PITCh vectors) The PITCh vector used was the pBApo-EF1α-pur vector, which contains the EF1α promoter (a forced expression promoter) and a puromycin resistance gene. The PITCh vector was constructed by inserting an annealed synthetic oligonucleotide into the pBApo-EF1α-pur vector. The pBApo-EF1α-pur vector was treated with BamHI and HindIII, subjected to electrophoresis, and then excised and purified. Subsequently, the annealed synthetic oligonucleotide and the pBApo-EF1α-pur vector treated with BamHI and HindIII were assembled using Ligation high Ver.2 (TOYOBO). The annealing method for the synthetic oligonucleotide was the same as described above. The base sequences of the sense oligonucleotide and antisense oligonucleotide contained in the inserted synthetic oligonucleotide are shown in SEQ ID NOs. 76 and 77, respectively.

[0091] 2. Creation of OVM-expressing cell lines using genome editing technology DF-1 cells were cultured at 37°C under 5% CO2 in KO-DMEM (Thermo Fisher Scientific) containing 10% FBS (Biological Industries) and 1×GlutaMAX (Thermo Fisher Scientific).

[0092] Immediately before gene transfer, DF-1 cells were placed in a 6-well culture plate at a rate of 1 × 10⁶ 6Cells were seeded to a specific cell / well ratio, and gene transduction was performed using lipofectamine 3000 (Thermo Fisher Scientific). The introduced plasmids were the three plasmids listed above (PITCh vector, pX330-OVM 5'-UTR, and pX330-EF1α donor), each administered at 0.8 μg for co-transduction. Drug selection with puromycin 1.0 μg / mL was performed for one week starting two days after gene transduction. After drug selection, the genome was extracted from the surviving cells using Puregene core kit A (QIAGEN), and PCR was performed to determine whether a cell population with the target mutation existed in the surviving cell population. Forward primer F2 was designed within the EF1α promoter derived from the PITCh vector, and reverse primer R2 was designed within the OVM gene itself in the DF-1 cell genome (F2 and R2 in Figure 19B). The nucleotide sequences of forward primer F2 and reverse primer R2 are shown in SEQ ID NOs. 78 and 79, respectively. The forward primer F2 and reverse primer R2 can be used to determine whether the 3' end of the inserted PITCh vector is inserted upstream of the OVM gene.

[0093] The obtained cells were converted into a monoclonal cell population by cloning at limiting dilution. Cells were prepared at a density of 50 cells per 96-well plate and seeded. After sufficient cell proliferation, the cells were harvested, and the genome was extracted using Puregene core kit A (QIAGEN). Genomic PCR was performed using forward primer F2 and reverse primer R2. In addition, genomic PCR was performed using forward primer F1 designed to be placed outside the inserted PITCh vector and reverse primer R1 designed to be placed inside the PITCh vector to determine whether the 5' end of the PITCh vector was also inserted (F1 and R1 in Figure 19B). The nucleotide sequences of forward primer F1 and reverse primer R1 are shown in SEQ ID NOs. 80 and 81, respectively. Furthermore, to determine whether the EF1α promoter was inserted in both alleles (homozygous) or in only one allele (heterozygous), forward primer F3 was designed to be placed outside the inserted PITCh vector (F3 in Figure 19B). The nucleotide sequence of forward primer F3 is shown in SEQ ID NO: 82.

[0094] As shown in Figures 20A and 20B, amplification of the target PCR product was confirmed in cell lines #2 and #3 obtained by cloning using primers designed for the region including the 5' and 3' junctions. Genotyping results shown in Figure 20C revealed that cell line #2 is heterozygous for the EF1α promoter, and cell line #3 is homozygous for the EF1α promoter.

[0095] 3. OVM expression analysis The expression analysis of the OVM gene using the inserted EF1α promoter was performed as follows using cell line #2 (EF1α-OVM heterozygous DF-1 cell line) and cell line #3 (EF1α-OVM homozygous DF-1 cell line).

[0096] (Detection of mRNA) RT-PCR was performed to confirm that the OVM gene was transcribed after the introduction of the EF1α promoter. RNA was extracted from wild-type DF-1 cells, cell line #2, and cell line #3, and cDNA was synthesized. RNA extraction and cDNA synthesis were performed using FastGene® RNA Premium Kit (Nippon Genetics Co., Ltd.) and SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Inc.), respectively, according to the protocol. RT-PCR was performed using the obtained cDNA as a template. The forward and reverse primers used in RT-PCR were designed for the 5' and 3' untranslated regions of the OVM mRNA, respectively. The nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs. 83 and 84.

[0097] Direct sequencing was performed using the PCR product as a template to determine whether the amplified PCR product matched a known nucleotide sequence. Cycle sequencing was performed using the SuperDye v3.1 Cycle Sequence Kit (AdvancedSeq), and analysis was performed using the SeqStudio® genetic analyzer (Thermo Fisher Scientific). As with the RT-PCR described above, the forward primer (sequence number 83) and the reverse primer (sequence number 84) were used.

[0098] As shown in Figure 21, RT-PCR confirmed at the mRNA level that the EF1α promoter promotes transcription of the OVM gene in DF-1 cells, leading to OVM gene expression. Direct sequencing revealed that the nucleotide sequences of OVM mRNA in cell lines #2 and #3 were almost identical to OVM transcription vriant 1 (NM_01308494.2) registered in NCBI, indicating that the amino acid sequence was not affected.

[0099] (Detection and quantification of OVM protein) Since OVM proteins are secreted proteins, they are secreted into the cell culture supernatant. By detecting and quantifying OVM proteins in a sample prepared from the culture supernatant, it is possible to confirm whether or not the OVM gene has been translated. Western blotting and sandwich ELISA methods using antibodies that specifically bind to OVM proteins can be used for the detection and quantification of OVM proteins.

[0100] For OVM protein detection, the culture supernatants of wild-type DF-1 cells, cell line #2, and cell line #3 were used as samples. Each DF-1 cell line was placed in a 6-well plate at a rate of 1 × 10⁶ 6 Cells were seeded in wells and cultured in FreeStyle® 293 Expression Mediums (Thermo Fisher Scientific). After one week, the culture supernatant was collected and concentrated for Western blotting. For sandwich ELISA, the culture supernatant was diluted 50-fold with PBS for the sample. An ULTRAFREE®-MC 5,000 NMWL Filter Unit (MILLIPORE) was used to concentrate the culture supernatant.

[0101] For the Western blotting method, 10 ng of purified OVM per lane was used as a positive control. NuPAGE® LDS Sample buffer (4×) (Thermo Fisher Scientific) was added to the prepared culture supernatant sample and heat-treated at 90°C for 10 minutes. SDS-PAGE was performed using this sample on a 15% polyacrylamide gel. For electrophoresis, a Compact PAGE (ATTO) in Tris-gly / PAGEL, High mode was used, with an electrophoresis time of 30 minutes. For the Western blotting method, an Immuno-Blot PVDF Membrane (BIORAD) treated with methanol for 10 minutes was used. SDS-PAGE-treated gels and methanol-treated Immuno-Blot PVDF membranes were blotted using blotting buffer (25 mM Tris, 192 mM glycine, 5% (v / v) methanol, 0.01% SDS) at 100 V, 0.25 A, and 1 hour. A PowerPac (BIORAD) was used for blotting.

[0102] After blotting, Immuno-Blot PVDF membranes were washed with 0.1% Tween20-PBS and blocked by standing overnight at 4°C with PVDF Blocking Reagent for Can Get Signal (TOYOBO). The blocked Immuno-Blot PVDF membranes were washed with 0.1% Tween20-PBS and then subjected to an antibody reaction using HRP-labeled anti-OVM mouse monoclonal antibody (prepared in our laboratory) diluted 3000-fold with Can Get Signal Solution 2 (TOYOBO). After the reaction, the membranes were washed with 0.1% Tween20-PBS and reacted with ECL® prime Western Blotting Detection Reagents (GE Healthcare), and detected using an Amersham Imager 680.

[0103] The sandwich ELISA method ensures quantitative detection by using two antibodies with different antigen epitopes. The OVM protein concentration in each culture supernatant sample prepared using the method described above was measured using the sandwich ELISA method. 100 μL / well of 0.5 μg / mL anti-OVM rabbit polyclonal antibody (prepared in our laboratory) was added as immobilized antibody to the ELISA plate and left to stand overnight at 4°C. 200 μL / well of 25% Block-Ace in PBS (DS Pharma Biomedical) was added for blocking and incubated at 37°C for 2 hours. Purified OVM was used as a positive control to create a calibration curve, starting from 100 ng / mL and serially diluted 2-fold. 10% Block-Ace in PBS was used for dilution, and 100 μL / well of each was added and incubated at 37°C for 1 hour. For detection, 100 μL / well of HRP-labeled 0.5 μg / mL anti-OVM mouse monoclonal antibody (prepared in our laboratory) was added and incubated at 37°C for 1 hour. The solution was then colored with a solution containing o-phenylenediamine (Nacalai Tesque) with the following composition. Citrate-phosphate buffer 5 mL o-Phenylenediamine 0.002g 1 μL of hydrogen peroxide

[0104] The solution was added at 50 μL / well and the reaction was carried out in the dark. The reaction was then stopped by adding 50 μL / well of 2M sulfuric acid, and the absorbance at 490 nm was measured using a MULTISCAN Sky (Thermo Fisher Scientific).

[0105] Figure 22 shows the results of the Western blot analysis. It was confirmed that the OVM gene in DF-1 cells was expressed at the protein level after knock-in of the EF1α promoter. Figure 23 shows the OVM concentration quantified by the sandwich ELISA method. The OVM concentration in the culture supernatant of cell line #3, which is homozygous for the EF1α promoter, was more than twice as high as that of cell line #2, which is heterozygous for the EF1α promoter, indicating that OVM protein expression is dependent on the EF1α promoter.

[0106] 3. Introduction and expression analysis of useful protein genes We investigated whether functional knockout of the OVM gene is possible by inserting a gene cassette to be expressed after the OVM start codon in OVM-expressing DF-1 cells. The gene cassette to be replaced included the hFGF2 gene and the marker gene (Zeo) as shown in Figure 24. R This includes the green fluorescent protein (EGFP). The gene cassette was inserted into the target OVM gene using the PITCh system with CRISPR / Cas9. The constructed PITCh vector and CRISPR / Cas9 vector will be referred to as PITCh-OVM and pX330-OVM below, respectively.

[0107] (Setting up pX330-OVM) The pX330-OVM was constructed using the pX330-U6-Chimeric BB-CBh-SpCas9 vector. It was prepared by inserting an annealed synthetic oligo, which serves as a gRNA template, into the pX330-U6-Chimeric BB-CBh-SpCas9 vector, which had been linearized with BpiI in the same manner as described above. The CRISPR / Cas9 used here targeted the nucleotide sequence shown underlined in Figure 24, downstream of the start codon of the OVM gene. The nucleotide sequences of the sense and antisense oligos contained in the inserted synthetic oligo are shown in SEQ ID NOs. 90 and 91, respectively.

[0108] (Building PITCh-OVM) In PITCh-OVM, the hFGF2 gene and a marker gene are inserted between microhomology sequences. As shown in FIG. 24, 16 bp immediately upstream of the start codon of the OVM gene and 21 bp including an intron from the end of exon 1 were used as the microhomology sequences. Additionally, the target sequence and PAM sequence of the pX330-OVM prepared as described above are added to the outside of the microhomology sequences, and both the genome and PITCh-OVM can be cleaved using one type of CRISPR / Cas9 vector. In-Fusion™ HD Cloning Kit (manufactured by TaKaRa) was used to prepare PITCh-OVM.

[0109] (Genome Editing of OVM-Expressing Cells) Genome editing was performed on cell line #2 and cell line #3 using the two types of vectors prepared above (PITCh-OVM and pX330-OVM). Cell line #2 and cell line #3 were seeded in 6-well culture plates at 1×10 6 cells / well immediately before gene transfer, and gene transduction was performed using lipofectamine 3000. Two days after gene transduction, drug selection with 200 μg / mL zeocin was performed for one week. After drug selection, genomic DNA was extracted from surviving cells using the Puregene core kit A (manufactured by QIAGEN), and PCR was performed to determine whether the surviving cell population contains a cell population having the target mutation. As shown in FIG. 25, by performing PCR using forward primer F4 designed for the EF1α promoter derived from the genome of OVM-expressing cells and reverse primer R3 designed for the PITCh-OVM vector, it can be determined whether PITCh-OVM is inserted into the allele where the EF1α promoter has been inserted. The nucleotide sequences of forward primer F4 and reverse primer R3 are set forth in SEQ ID NOs: 92 and 93, respectively.

[0110] The obtained cells were cloned using limiting dilution to form a monoclonal cell population. The cells were prepared to 50 cells per 96-well plate and seeded. After sufficient cell proliferation, the cells were harvested, and the genome was extracted using Puregene core kit A (QIAGEN). Genomic PCR was performed using forward primer F4 and reverse primer R3. Furthermore, as shown in Figure 25, genomic PCR was performed using forward primer F5 designed for the inserted PITC-OVM and reverse primer R4 designed for the OVM gene on the 3' side of PITCh-OVM to determine whether the insertion was accurate on the 3' side of PITCh-OVM. The nucleotide sequences of forward primer F5 and reverse primer R4 are shown in SEQ ID NOs. 94 and 95, respectively. Furthermore, to confirm that the full length of PITCh-OVM is inserted into the allele containing the EF1α promoter, PCR is performed using forward primer F2 and reverse primer R5, designed for the 3' OVM gene of PITC-OVM as shown in Figure 25. This allows determination of whether PITCh-OVM is homozygous (inserted into both alleles) or heterozygous (inserted into only one allele) in cell line #3, which is homozygous for the EF1α promoter. The nucleotide sequence of reverse primer R5 is shown in SEQ ID NO: 96.

[0111] As shown in Figures 26A and 26B, amplification of the target PCR product was confirmed in cell lines #2-3 and #3-4 obtained by cloning using primers designed for the region including the 5' and 3' junctions. Genotyping results shown in Figure 26C confirmed the insertion of the hFGF2 cassette in cell lines #2-3 and #3-4.

[0112] (Detection and quantification of proteins) Cell lines #2-3 and #3-4 each have PITCh-OVM inserted into the OVM gene of the allele in which the EF1α promoter is inserted. We investigated whether the OVM gene was functionally knocked out and whether the ability to secrete OVM protein was lost in these cell lines using the culture supernatant of each cell line. Since OVM protein is a secreted protein, OVM protein is secreted into the cell culture supernatant in cell lines #2 and #3, which are OVM-expressing cell lines. Therefore, whether the modification to the OVM gene contributes to functional knockout can be confirmed by performing Western blotting and sandwich ELISA using the cell culture supernatant.

[0113] Similarly to the above, OVM protein in the culture supernatant of heterozygous cell lines #2-3 and homozygous cell lines #3-4 was detected and quantified by Western blotting and sandwich ELISA. As shown in Figure 27, Western blotting confirmed that OVM protein was not secreted into the culture supernatant of cell lines #2-3 and #3-4. In the sandwich ELISA, as shown in Figure 28, the concentration of OVM protein in the culture supernatant of cell lines #2-3 and #3-4 was below the detection limit, similar to wild-type DF-1. Therefore, it was shown that the OVM gene was knocked out in cell lines #2-3 and #3-4 by knocking in PITCh-OVM.

[0114] [Example 3: Genome editing of the OVA gene locus] 1. Development of genome editing tools In this example, similar to the knock-in of the hFGF2 gene between exon 1 and exon 2 of the OVA locus shown in Example 1, the hFGF2 gene was knocked in downstream of exon 8. In this example, the region encoding T2A as a 2A peptide was inserted between the OVA gene and the hFGF2 gene. Homologous recombination (HR) was used for the knock-in downstream of exon 8 of the OVA locus. The genome editing tools used for the HR knock-in were two TALEN expression vectors (Left and Right) targeting the OVA locus and a donor vector containing the hFGF2 gene.

[0115] (Preparation of TALEN expression vectors) Based on the target sequence of exon 8 of the OVA locus shown in Figure 29, two types of TALEN expression vectors were constructed.

[0116] (Construction of a donor vector carrying the hFGF2 gene) Figure 30 shows a vector map of the pCR2.1(trademark)-TOPO(trademark)TA vector (manufactured by Thermo Fisher Scientific) used to prepare the donor vector. First, using the specified primers, approximately 1000 bp around the target sequence of the OVA gene locus was cloned into the pCR2.1(trademark)-TOPO(trademark)TA vector as a homologous sequence. Subsequently, T2A-hFGF2-Puro R The product was amplified using a predetermined primer, and after an infusion reaction, it was transformed into competent cells. Furthermore, Puro R to Neo R To modify the cells, the In Fusion reaction was performed again, and the cells were transformed into competent cells. After small culture, the plasmid was extracted, treated with BglII, and chained. Figure 31 shows a map of the donor vector before treatment with BglII. Table 3 shows the nucleotide sequences of the primers used to prepare the donor vector.

[0117] [Table 3]

[0118] 2. Introduction of useful protein genes The constructed genome editing tool was introduced into the EF1α-Exon1 cell strain obtained in Example 1, and drug selection with 100 μg / mL neomycin was performed for two weeks. Genomic DNA was extracted from polyclonal cells and subjected to genomic PCR. The positions of forward primer P10 and reverse primer P11 for hFGF2 gene knock-in detection are shown in Figure 32B. The nucleotide sequences of forward primer P10 and reverse primer P11 are shown in Sequence ID No. 113 and 114, respectively. Subsequently, the cells were monocloned using the limiting dilution method and subjected to genomic PCR.

[0119] As shown in Figure 33, we confirmed that the hFGF2 gene was knocked into exon 8 of the OVA locus in clones #1 and #8. Furthermore, to investigate whether the hFGF2 gene was knocked into downstream of the EF1α promoter present in one allele, we performed genomic PCR using forward primer P12 and reverse primer P13, whose positions are shown in Figure 32B. The nucleotide sequences of P12 and P13 are shown in SEQ ID NOs. 115 and 116. As shown in Figure 34, among clones #1 and #8 in which hFGF2 gene knock-in was confirmed, knock-in downstream of the EF1α promoter was confirmed in clone #8. When clone #8 was subjected to sequencing analysis, it was confirmed that the hFGF2 gene was knocked into the target position of the OVA locus.

[0120] Similar to clone number #8, knock-in of the hFGF2 gene at the target position of the OVA locus was confirmed in clone numbers #23 and #26. Three strains (clones #8, #23, and #26) were cultured, RNA was extracted, and cDNA was synthesized. RT-PCR was performed using the synthesized cDNA. The predicted sequences after splicing in the three strains are shown in Figure 32C. The size of the augmented byproducts by RT-PCR using forward primers (Fwd2) and reverse primers (Rev2), whose nucleotide sequences are shown in SEQ ID NOs. 117 and 118, respectively, was 613 bp. As a result of RT-PCR, as shown in Figure 35, it was confirmed that the OVA gene and the hFGF2 gene were transcribed in clone numbers #8, #23, and #26.

[0121] An ELISA method was performed to determine whether OVA protein and hFGF2 protein were expressed in clones #8, #23, and #26. For OVA protein detection, the culture supernatant from clones #8, #23, and #26 cultured for 7 days was used. For FGF2 protein detection, lysate samples from clones #8, #23, and #26 were used. Lysate samples were prepared according to the Human FGF2 sandwich ELISA kit (Proteintech). Figures 36 and 37 show the concentrations of OVA protein and hFGF2 protein, respectively. OVA protein was present in the culture supernatant at concentrations ranging from 80 ng / ml to 390 ng / ml. hFGF2 protein was present in the lysate at concentrations ranging from 520 pg / ml to 1500 pg / ml.

[0122] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention. [Industrial applicability]

[0123] This invention is suitable for evaluation systems for bioreactors and for the construction of bioreactors. [Explanation of Symbols]

[0124] 1,2 Marker genes 3,4 Intraocular protein gene loci 5,6 Foreign promoter sequence 7,8 Foreign genes

Claims

1. The genome has a first exogenous promoter sequence that causes constitutive expression of at least one gene contained in the ovalbumin gene locus, The aforementioned gene is, Contains the ovalbumin gene, The first foreign promoter sequence is, Located upstream of exon 1 of the ovalbumin gene, A cultured cell line of birds.

2. The gene is The invention further includes an exogenous gene knocked in upstream or downstream of the open reading frame of the ovalbumin gene at the ovalbumin gene locus, The cultured avian cell line according to claim 1.

3. The first foreign promoter sequence is, The EF1α promoter sequence is A cultured avian cell line according to claim 1 or 2.

4. The genome further comprises a second exogenous promoter sequence that causes constitutive expression of at least one gene contained in the ovomucoid locus, The gene constitutively expressed by the first foreign promoter sequence is The present invention further includes a first foreign gene knocked in upstream or downstream of the open reading frame of the ovalbumin gene at the ovalbumin locus, The gene constitutively expressed by the second exogenous promoter sequence is A second foreign gene knocked in upstream of the ovomucoid gene, The second exogenous promoter sequence is, Located upstream of the second foreign gene, The cultured avian cell line according to claim 1.

5. These are genetically modified DF-1 cells. A cultured avian cell line according to claim 1 or 2.

6. The procedure involves knocking in an exogenous promoter sequence that constitutively expresses at least one gene contained in the ovalbumin gene locus upstream of exon 1 of the ovalbumin gene into the genome of a cultured avian cell line, and The steps include knocking in an exogenous gene upstream or downstream of the open reading frame of the ovalbumin gene at the ovalbumin gene locus, Includes, The aforementioned gene is, Including the ovalbumin gene and the aforementioned foreign gene, A method for producing cultured cell lines for evaluation of bioreactors.

7. A cultured avian cell line according to claim 1, A reagent for knocking in an exogenous gene as the gene upstream or downstream of the open reading frame of the ovalbumin gene at the ovalbumin gene locus, A bioreactor evaluation kit equipped with the necessary components.

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