Method for improving efficiency of avian egg bioreactor

Abstract

Provided are: a method for increasing the expression of a foreign protein, the method comprising expressing, in a poultry cell, a construct containing a 3' untranslated region of a gene encoding a foreign protein and a gene encoding an oviduct-specific protein, wherein the 3' untranslated region of the gene encoding an oviduct-specific protein is placed on the 3' side of the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon; poultry knocked-in by placing a construct including a 3' untranslated region of a gene encoding a foreign protein and a gene encoding an oviduct-specific protein in an oviduct-specific locus, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is placed on the 3' side of the gene encoding the foreign protein and the gene encoding the foreign protein contains a stop codon; and eggs laid by the poultry.

Description

Methods for improving the efficiency of avian egg bioreactors

[0001] RELATED APPLICATIONS This application claims the benefit of priority from Application No. 2024-072407, filed with the Japan Patent Office on April 26, 2024. The priority application is hereby incorporated by reference in its entirety.

[0002] The present invention relates to genetic recombination technology. In particular, the present invention relates to a method for increasing expression of a foreign gene in avian cells, avian individuals, and avian eggs.

[0003] In recent years, so-called "animal factories," which use genetic engineering techniques to produce useful substances in animals, have been attracting attention. One such technology, the egg bioreactor, is a technology for mass-producing foreign proteins in egg whites by genetically modifying chickens (Patent Document 1). In female genetically modified chickens, foreign proteins are expressed in cells of the oviduct tissue, which are secreted extracellularly along with endogenous egg white proteins and accumulate in the lumen of the oviduct tissue together with egg white proteins, ultimately obtaining the recombinant protein in the egg white of chicken eggs. The greater the amount of foreign protein accumulated in the egg white, the more useful the production system. Therefore, there is a need for a technology that can more efficiently express foreign proteins in cells of the oviduct tissue and produce them at high concentrations in the egg whites of recombinant birds.

[0004] Known methods for highly efficient expression of foreign proteins in cells include using strong promoters, optimizing codons to suit the host, and modifying the 5' and 3' untranslated regions. However, because gene expression control mechanisms differ depending on the host, there are few common, highly efficient expression techniques. Furthermore, when expressing foreign proteins in animal factories, it is necessary to make the expression site tissue-specific to avoid affecting the development and health of individual animals.

[0005] In particular, there are far fewer reports on the production of foreign proteins using avian cells or avian individuals than there are for other species, and there has been almost no research into techniques for improving the efficiency of foreign protein expression.

[0006] It has been previously known that when expressing a foreign protein in the egg white of poultry, the foreign protein can be expressed with high efficiency by inserting a foreign gene under the control of a promoter of the egg white protein, and that this allows a large amount of foreign protein to accumulate in the egg white of the recombinant individual (Patent Document 1). However, no technique has been identified that improves on this technique or uses a different method to express foreign proteins with higher efficiency.

[0007] International Application Publication WO2017 / 111144A1

[0008] There is a need for techniques that further improve the efficiency of recombinant protein expression in avian cells, avian individuals, and avian eggs.

[0009] To solve the above problems, the inventors conducted various studies and developed a technology for high expression of foreign proteins in avian oviduct cells. In the process, they made improvements to the state of the transgene, and then introduced gene vectors before and after the improvements into avian individuals and avian cells to express the same protein, comparing the results to examine the effects of the improvements. Specifically, they established and compared knock-in chickens in which 650 bases of the 3' untranslated region of ovalbumin mRNA were added 3' to the stop codon of the foreign gene with those in which they were not (conventional knock-in chickens). As a result, they found that foreign gene expression and foreign protein accumulation were increased in eggs from chickens in which 650 bases of the 3' untranslated region of ovalbumin mRNA were added 3' to the stop codon of the foreign gene, leading to the completion of the present invention.

[0010] That is, the present invention provides the following: (1) A method for increasing expression of a foreign protein, comprising expressing in an avian cell a construct comprising a gene encoding a foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein, wherein the 3' untranslated region of the gene encoding the foreign protein is located 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein comprises a stop codon. (2) The method according to (1), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 9. (3) The method according to (1) or (2), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 75% or more identity to the nucleotide sequence shown in SEQ ID NO: 25. (4) The method according to any of (1) to (3), wherein the avian cell is an oviduct-specific cell. (5) A method for producing a bird that expresses a foreign protein, comprising knocking in a construct comprising a gene encoding a foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein by locating it at an oviduct-specific locus, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is located on the 3' side of the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon. (6) The method according to (5), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 9. (7) The method according to (5) or (6), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 75% or more identity to the nucleotide sequence shown in SEQ ID NO: 25. (8) The method according to any of (5) to (7), wherein the oviduct-specific locus is the ovalbumin locus.(9) A bird in which a construct comprising the 3' untranslated region of a gene encoding a foreign protein and a gene encoding an oviduct-specific protein has been knocked in by locating the construct at an oviduct-specific locus, the 3' untranslated region of the gene encoding the foreign protein being located on the 3' side of the gene encoding the foreign protein, and the gene encoding the foreign protein containing a stop codon. (10) The bird according to (9), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 9. (11) The bird according to (9) or (10), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 75% or more identity to the nucleotide sequence shown in SEQ ID NO: 25. (12) The bird according to any of (9) to (11), wherein the oviduct-specific locus is the ovalbumin locus. (13) A construct comprising a gene encoding a foreign protein and a 3' untranslated region of a gene encoding an oviduct-specific protein, wherein the 3' untranslated region of the gene encoding the foreign protein is located 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein comprises a stop codon. (14) The construct according to (13), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein comprises a nucleotide sequence having 85% or more identity to the nucleotide sequence shown in SEQ ID NO: 9. (15) The construct according to (13) or (14), wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein comprises a nucleotide sequence having 75% or more identity to the nucleotide sequence shown in SEQ ID NO: 25. (16) An expression vector comprising the construct according to any one of (13) to (15). (17) An avian cell comprising the construct according to any one of (13) to (15) or the expression vector according to claim 16. (18) A method for producing a foreign protein, comprising culturing the cell according to (17). (19) An egg laid by the bird according to any one of (9) to (12). (20) A method for producing a foreign protein, comprising obtaining the foreign protein from the egg according to (19).

[0011] According to the present invention, the expression efficiency of foreign proteins in avian cells, avian individuals, and avian eggs can be improved. The present invention can be used to improve the protein production efficiency in, for example, a chicken egg bioreactor.

[0012] FIG. 1 is a schematic diagram of constructs (conventional and improved (constructs of the present invention)) containing a foreign gene (canine IL-4) used in the experiments of Example 1. FIG. 2 is a schematic diagram of donor constructs (conventional and improved (constructs of the present invention)) prepared in Example 1 for chicken genetic recombination (gene knock-in). FIG. 3 is a schematic diagram showing the insertion of a donor construct into exon 2 of the chicken ovalbumin gene. FIG. 4 shows the results of Coomassie brilliant blue staining comparing the expression levels of proteins contained in the egg white of eggs laid by chickens into which the construct of the present invention was introduced and in the egg white of eggs laid by chickens into which the conventional construct was introduced. FIG. 5 is a schematic diagram of constructs (conventional and improved (constructs of the present invention)) prepared in Example 1 for expressing a foreign gene in poultry oviduct cells.

[0013] In one aspect, the present invention provides a method for increasing expression of a foreign protein, comprising expressing in an avian cell a construct comprising a gene encoding the foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein, wherein the 3' untranslated region of the gene encoding the foreign protein is located 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

[0014] As used herein, a construct refers to an artificially produced polynucleotide. The construct may or may not be in the form of a vector. When the construct is not in the form of a vector (for example, in the form of a cassette), it is usually incorporated into a vector for use. The construct may be a single-stranded nucleic acid or a double-stranded nucleic acid. The construct herein comprises a gene encoding a foreign protein and the 3' untranslated region of a gene encoding a fallopian tube-specific protein. In the construct, the 3' untranslated region of the gene encoding the fallopian tube-specific protein is located 3' from the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

[0015] The construct comprises a gene encoding a foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein. In the construct, the 3' untranslated region of the gene encoding the oviduct-specific protein is located 3' to the gene encoding the foreign protein. The gene encoding the foreign protein contains a stop codon.

[0016] The construct may contain, on the 5' side of the gene encoding the foreign protein, any or desired sequence, for example, a nucleotide sequence encoding a promoter, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a Flag epitope tag, a nucleotide sequence encoding a protease cleavage sequence such as an HRV 3C sequence or a TEV protease cleavage sequence, etc. One or more of these any or desired sequences may be contained in the vector into which the construct is to be incorporated.

[0017] The construct may include any or desired sequence, such as a base sequence encoding a linker, a base sequence encoding a protease cleavage site, or a base sequence encoding a tag for purification such as a histidine tag, between the stop codon of the gene encoding the foreign protein and the gene encoding the 3' untranslated region of the gene encoding the oviduct-specific protein.

[0018] The construct may contain any or desired sequence, such as a terminator sequence for terminating transcription of SV40, hGH, BGH, rbGlob, etc., a gene encoding a fluorescent protein such as the GFP gene, a drug resistance marker gene such as puromycin or neomycin, etc., on the 3' side of the gene encoding the 3' untranslated region of the gene encoding the fallopian tube-specific protein. One or more of these any or desired sequences may be contained in a vector into which the construct is to be incorporated.

[0019] The foreign protein may be any type of protein. Examples of foreign proteins include, but are not limited to, antibodies or fragments thereof (e.g., scFv, Fab, Fab', F(ab')2, Fv, single-chain antibodies, scFv, dsFv), enzymes, hormones, growth factors, cytokines, interferons, collagen, extracellular matrix molecules, functional polypeptides such as vaccines, agonistic proteins, and antagonistic proteins. The foreign protein may be a natural protein or a mutant or fragment thereof. The foreign protein may be an artificial protein. When the foreign protein is a physiologically active protein that can be used as a pharmaceutical for administration to humans, it is derived from a mammal, preferably a human.

[0020] The construct may contain one or more genes encoding foreign proteins. The genes encoding foreign proteins may be single-stranded or double-stranded nucleic acids. When multiple genes encoding foreign proteins are present, it is sufficient that the multiple genes are expressed under the control of the oviduct-specific gene. For example, multiple genes may be expressed via an IRES or other sequence. Alternatively, multiple proteins may be expressed simultaneously under the control of the ovalbumin promoter via an IRES or other sequence encoding the 2A peptide, and the peptide may be expressed in a cleaved form. The codon usage of the gene encoding the foreign protein may be changed to facilitate expression in avian cells.

[0021] Avian oviduct-specific proteins are known, and include, but are not limited to, ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, lysozyme, and the like.

[0022] The 3' untranslated regions of genes encoding various oviduct-specific proteins are known. For example, the nucleotide sequence of the 3' untranslated region of the chicken ovalbumin gene is shown in SEQ ID NO: 9, the nucleotide sequence of the 3' untranslated region of the quail ovalbumin gene is shown in SEQ ID NO: 21 (88% sequence identity to SEQ ID NO: 9 in BLAST), the nucleotide sequence of the 3' untranslated region of the turkey ovalbumin gene is shown in SEQ ID NO: 22 (92% sequence identity to SEQ ID NO: 9 in BLAST), and the nucleotide sequence of the 3' untranslated region of the ptarmigan ovalbumin gene is shown in SEQ ID NO: 23 (90% sequence identity to SEQ ID NO: 9 in BLAST).

[0023] The construct may contain one or more genes encoding the 3' untranslated region of the gene encoding the oviduct-specific protein (for example, arranged in tandem).

[0024] The nucleotide sequence of the gene encoding the 3' untranslated region of the gene encoding the oviduct-specific protein may be any sequence that can increase expression of a foreign protein. For example, based on the wild-type nucleotide sequence (SEQ ID NO: 9) (650 nucleotides) of the 3' untranslated region of the chicken ovalbumin gene, the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein may include a nucleotide sequence that has, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID NO: 9.

[0025] The region from bases 250 to 650 (SEQ ID NO: 25) (401 bases) of the nucleotide sequence (SEQ ID NO: 9) of the 3' untranslated region of the chicken ovalbumin gene is highly conserved and homologous to corresponding regions in many birds, and this region is believed to be functional. For example, the 3' untranslated region of the kiwi ovalbumin gene (SEQ ID NO: 24) has 78% identity to the nucleotide sequence of SEQ ID NO: 25 in BLAST analysis. Therefore, a gene containing a nucleotide sequence that is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identical to the nucleotide sequence of SEQ ID NO: 25 may be positioned 3' to the gene encoding the foreign protein as a gene encoding the 3' untranslated region of the gene encoding the oviduct-specific protein.

[0026] The identity of the nucleotide sequences can be determined by known methods, for example, using BLAST.

[0027] The nucleotide sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein may be one that hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 9 or 25. Stringent conditions are known, and examples thereof include the following: 0.25M Na ２ HPO ４ The hybridization is carried out for 16 to 24 hours in a buffer solution containing 1× Denhardt's solution, pH 7.2, 7% SDS, 1 mM EDTA, and 1× Denhardt's solution at a temperature of 60 to 68° C., preferably 65° C., and more preferably 68° C., and further in a buffer solution containing 20 mM Na ２ HPO ４, pH 7.2, 1% SDS, 1 mM EDTA, followed by two 15-minute washes at 60 to 68°C, preferably 65°C, and more preferably 68°C; or, alternatively, overnight prehybridization at 42°C in a hybridization solution containing 25% formamide, or for more stringent conditions, 50% formamide, 4x SSC (sodium chloride / sodium citrate), 50 mM HEPES pH 7.0, 10x Denhardt's solution, and 20 µg / ml denatured salmon sperm DNA, followed by washing at 37°C in a buffer containing 1x SSC and 0.1% SDS, or for more stringent conditions, at 42°C in a buffer containing 0.5x SSC and 0.1% SDS, or for even more stringent conditions, at 65°C in a buffer containing 0.2x SSC and 0.1% SDS. Needless to say, stringent conditions are not limited to the above examples.

[0028] Furthermore, the nucleotide sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein may comprise a sequence in which 1-200, for example, 1-150, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 nucleotides have been substituted, added, inserted, or deleted in the nucleotide sequence shown in SEQ ID NO: 9. Furthermore, the nucleotide sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein may comprise a sequence in which 1-120, for example, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 nucleotides have been substituted, added, inserted, or deleted in the nucleotide sequence shown in SEQ ID NO: 25.

[0029] The increase in expression of the gene encoding the foreign protein by the method of the present invention is 1.5-fold or more, preferably 2-fold or more, more preferably 3-fold or more, and even more preferably 5-fold or more, compared to when the 3' untranslated region of the gene encoding the fallopian tube-specific protein is not added. The increase in production of the foreign protein by the method of the present invention is 1.5-fold or more, preferably 2-fold or more, more preferably 3-fold or more, and even more preferably 5-fold or more, compared to when the 3' untranslated region of the gene encoding the fallopian tube-specific protein or a mutant thereof is not added. The expression level of the foreign gene and the production level of the foreign protein can be determined using known methods, for example, by real-time PCR, measuring the fluorescence intensity of a fluorescent protein, or by subjecting a sample to polyacrylamide gel electrophoresis, staining with Coomassie Brilliant Blue, and measuring the signal intensity of the stained band using image analysis software.

[0030] Expression of a construct in avian cells can be achieved by introducing the construct as a vector into the avian cells, or by introducing a vector containing the construct into the avian cells. Various vectors are known, with expression vectors being preferred. Examples of expression vectors include, but are not limited to, pCAG vector, pCMV vector, and pEF1α vector. The construct or vector can be introduced into avian cells by any method as long as the desired foreign protein is expressed. Many introduction methods are known, including, but not limited to, microinjection, lipofection, electroporation, or viral vector infection.

[0031] Avian cells may be derived from any type of bird, including, but not limited to, poultry, such as chickens, quails, turkeys, ducks, geese, cockroaches, bantams, pigeons, ostriches, pheasants, guinea fowl, and kiwis.

[0032] The avian cells may be derived from any part of a bird. The avian cells may be cultured cells. The avian cells may be oviduct cells, preferably primary oviduct cells. When expressing a foreign protein in cells other than oviduct cells, the construct may contain a promoter for expressing the foreign gene, or the vector into which the construct is incorporated may contain such a promoter. When expressing a foreign protein in oviduct cells, the construct or vector may not contain a promoter for expressing the foreign gene. For example, when introducing a construct into oviduct cells, the construct may be positioned downstream of the promoter of a gene encoding an oviduct-specific protein. Promoters are known and can be selected appropriately by those skilled in the art. The promoter may be, for example, a promoter of an oviduct-specific gene (such as the promoter of the ovalbumin gene). It is preferable that the gene encoding the foreign protein is positioned in frame under the control of the promoter.

[0033] In a further aspect, the present invention provides an expression vector comprising the above-described construct.In a further aspect, the present invention provides an avian cell comprising the above-described construct or an expression vector comprising the above-described construct.

[0034] In a further aspect, the present invention provides a method for producing a foreign protein, which comprises culturing an avian cell containing the above-mentioned construct or a vector containing the above-mentioned construct.

[0035] Culturing methods and conditions for avian cells are known to those skilled in the art and can be appropriately selected and modified depending on the type of avian cell, the type of foreign protein, the structure of the construct or vector, and the like. The foreign protein may be produced intracellularly or extracellularly. The produced foreign protein can be purified and / or isolated using known methods. For example, foreign proteins can be purified and / or isolated using techniques such as sonication, solubilization using a solubilizing agent such as arginine hydrochloride, ammonium sulfate fractionation, chromatography such as ion chromatography, gel filtration chromatography, hydrophobic interaction chromatography, and affinity chromatography, lyophilization, crystallization, cleavage or removal of linkers with proteases, peptidases, or drugs, and the like. The foreign protein may be obtained as a single protein or as a fusion protein.

[0036] In another aspect, the present invention provides a method for producing a bird that expresses a foreign protein, comprising knocking in a construct comprising a gene encoding the foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein by placing it into an oviduct-specific gene locus, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is placed 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

[0037] In another aspect, the present invention provides a bird that has been knocked in by placing a construct comprising a gene encoding a foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein into an oviduct-specific gene locus, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is located 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

[0038] The method for producing the birds is described below (see also International Application Publication WO2017 / 111144A1, which is incorporated herein by reference). The method for producing the birds is not limited to the method described below.

[0039] In the method for producing birds, a construct is knocked into avian cells, preferably into avian primordial germ cells. Knock-in can be performed by known methods. For example, knock-in may be performed using genome editing. Preferably, the construct is placed at an oviduct-specific gene locus. Oviduct-specific genes are known, and examples include, but are not limited to, genes encoding ovalbumin, ovomucoid, ovotransferrin, ovoglobulin, or lysozyme. A foreign gene is preferably placed so that it is expressed under the control of the oviduct-specific gene. Preferably, the placement is in-frame with the oviduct-specific gene. Furthermore, it is preferable to align the translation start point of the foreign gene with the translation start point of the oviduct-specific gene. When aligning the translation start point of the foreign gene with the translation start point of the oviduct-specific gene, a genetic sequence surrounding the translation start point of the oviduct-specific gene may be inserted immediately before the translation start point of the foreign gene. Another preferred placement may be one in which the translation start point of the construct is located 5' to the translation start point of the oviduct-specific gene.

[0040] In one specific example, when the fallopian tube-specific gene is the ovalbumin gene and a foreign gene is inserted under the control of its promoter, the construct may be knocked in so that the 5' end of the foreign gene is located in exon 2 of the gene.

[0041] The gene encoding the foreign protein in the construct may be a single-stranded nucleic acid or a double-stranded nucleic acid. In the case of a double-stranded nucleic acid, it may be introduced in the form of a plasmid vector, a BAC (bacterial artificial chromosome) vector, or the like.

[0042] In one specific example, primordial germ cells into which the construct has been knocked in are transplanted into recipient embryos to produce chimeric knock-in birds, and birds in which the construct has been knocked in are obtained in their progeny. In the knock-in individuals, an expression product of the construct containing the foreign protein of interest is secreted from the oviduct cells instead of an expression product of the oviduct-specific gene. The secreted expression product accumulates in the oviduct lumen and is contained in the egg white. As a result, eggs laid by the birds contain the expression product of the construct.

[0043] Therefore, in a further aspect, the present invention provides eggs laid by birds into which the construct has been knocked in. The eggs contain at least 1.5 times, preferably at least 2 times, more preferably at least 3 times, and even more preferably at least 5 times the amount of foreign protein as compared to eggs laid by birds into which the construct has not been knocked in.

[0044] A specific example of such birds includes, but is not limited to, birds in which the construct is placed in frame with the ovalbumin gene under the control of the ovalbumin promoter in oviduct cells.

[0045] The genotype of the knocked-in construct (gene) may be heterozygous (+ / -) or homozygous (+ / +). Eggs laid by females homozygous for the knocked-in gene contain more of the foreign protein of interest than eggs laid by females heterozygous for the knocked-in gene.

[0046] In a further aspect, the present invention provides a method for producing a foreign protein, comprising obtaining the foreign protein from eggs laid by the above-mentioned birds. Egg white containing the foreign protein may be obtained, and the foreign protein may be purified and / or isolated from the egg white. Purification and / or isolation of the foreign protein is as described above.

[0047] In a further aspect, the present invention provides a construct comprising a gene encoding a foreign protein and a 3' untranslated region of a gene encoding a fallopian tube-specific protein, wherein the 3' untranslated region of the gene encoding the fallopian tube-specific protein is located 3' from the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon. The construct is as described above.

[0048] The present invention will be explained in more detail and specifically below by showing examples, but the examples should not be construed as limiting the present invention.

[0049] Example 1: Generation of gene knock-in chickens using canine IL-4 as a model and increasing the efficiency of foreign protein expression in egg white (1) Constructs containing a canine IL-4 expression gene (knock-in donor gene) Figure 1 shows schematic diagrams of the constructs (conventional and improved (constructs of the present invention)) containing a foreign gene (canine IL-4) used in the experiment. DNA (SEQ ID NO: 1) encoding the ovotransferrin signal peptide and the four amino acids following it (amino acids 1-23 of the ovotransferrin immature protein, sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 29)) was used as the common signal peptide. Ligated to the 3' side of this sequence were DNA (SEQ ID NO: 2) encoding a 3xFlag epitope tag (sequence DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 30)), DNA (SEQ ID NO: 3) encoding a TEV protease cleavage sequence (ENLYFQG (SEQ ID NO: 31)), and DNA (SEQ ID NO: 4) encoding a canine IL-4 mature protein. Ligated to the 3' side were DNA (SEQ ID NO: 5) encoding a TEV protease cleavage sequence and DNA (SEQ ID NO: 6) encoding a 6xHis epitope tag, and a termination codon (TGA) was positioned. A terminator sequence (BGH terminator sequence) (SEQ ID NO: 7) for terminating transcription was positioned 3' to the termination codon. This is schematically shown as a conventional type in the upper panel of Figure 1. The entire DNA sequence, i.e., the DNA encoding the foreign gene ovotransferrin signal peptide-3xFlag-TEV cleavage sequence-canine IL-4-TEV cleavage sequence-6xHis, followed by the terminator sequence, is shown in SEQ ID NO: 8. In the present invention, a foreign gene was prepared in which 650 bases of the 3'-untranslated region of chicken ovalbumin (SEQ ID NO: 9) were placed on the 3' side of the foreign gene. This improved version is shown schematically in the lower panel of Figure 1, and the entire sequence is shown in SEQ ID NO: 10.

[0050] Next, the conventional and improved genes were incorporated into chickens by genetic recombination, and the foreign protein was expressed and secreted in the oviduct cells of the chickens under the control of the ovalbumin promoter, thereby expressing the foreign protein in egg white. The effects of the present invention were then verified by comparing the two constructs. A schematic diagram of the donor constructs (conventional and improved (constructs of the present invention)) prepared for chicken genetic recombination (gene knock-in) is shown in Figure 2. A canine IL-4 gene unit was ligated as a conventional or improved foreign gene to the 3' side of the 2.8 kb upstream of the ovalbumin translation initiation site (SEQ ID NO: 11), and further 3' to that, an SV40 promoter, a neomycin resistance gene (SEQ ID NO: 12), and a 3.1 kb gene sequence from ovalbumin exon 2 onward (SEQ ID NO: 13) were placed. The entire sequences were those of the conventional IL-4 donor (SEQ ID NO: 14) and the improved IL-4 donor (SEQ ID NO: 15), and these were inserted into the plasmid pBlue Script II (SK+) (Stratagene, USA, now Agilent Technologies) to give pBS-dIL-4 donor (conventional) and pBS-dIL-4-OVA3UTR donor (improved), respectively.

[0051] (2) Genome editing using chicken male primordial germ cells and production of knock-in chickens Knock-in to the ovalbumin locus was performed using a chicken male primordial germ cell line in accordance with International Application Publication WO2017 / 111144A1, targeting the ovalbumin gene using the CRISPR method. OligoDNAs represented by SEQ ID NOs: 18 and 19 were synthesized targeting SEQ ID NO: 17 (OVATg1) in the sequence (SEQ ID NO: 16) containing the translation start point of exon 2 of the ovalbumin gene shown in Figure 3, and the 5'-ends were phosphorylated using T4 Polynucleotide Kinase. The mixture was then heated to 98 ° C. and annealed by slowly cooling to room temperature. This DNA fragment was inserted into the BbsI cleavage site of the plasmid px330-Puror (px330-Puror-OVATg1), which had been prepared by inserting the puromycin resistance gene unit of SEQ ID NO: 20 into the NotI site of the plasmid px330 (AddGENE, USA).

[0052] The above-mentioned gene (plasmid) was introduced into a chicken male primordial germ cell line (prepared in accordance with Nature. 2006 Jun 8;441(7094):766-769. doi: 10.1038 / nature04831.) collected from the blood of a White Leghorn male embryo. ５ ~5 x 10 ５ Male primordial germ cell lines were washed with PBS, suspended in OPTI-MEM, and transfected with 0.8 μg of px330-Puror-OVATg1 and 0.8 μg of pBS-dIL-4 donor or 0.8 μg of pBS-dIL-4-OVA3UTR donor using 3 μl of Lipofectamine 2000. More specifically, Lipofectamine 2000 and the plasmid were mixed in 80 μl of OPTI-MEM, mixed with the male primordial germ cell line, and then allowed to stand at room temperature for approximately 5 minutes. 500 μl of antibiotic-free medium was then added, and the mixture was allowed to stand at 37°C for approximately 1 to 4 hours before being plated on feeder cells. From day 3 after transfection, neomycin (G418 disulfate, Nacalai Tesque, Japan) was added to a final concentration of 0.5 mg / ml. The medium was exchanged as appropriate, and cells growing in the presence of neomycin at a final concentration of 0.5 mg / ml were collected and used as dIL-4 donor (conventional) knock-in primordial germ cells and dIL-4-OVA3UTR donor (improved) knock-in primordial germ cells, respectively.

[0053] The dIL-4 donor (conventional) knock-in primordial germ cells and the dIL-4-OVA3UTR donor (improved) knock-in primordial germ cells were transplanted into the blood of 2.5-day-old White Leghorn embryos (recipient embryos) by microinjection. Prior to transplantation, the fertilized eggs were irradiated with 5 or 6 Gy of ionizing radiation before incubation to reduce the number of endogenous primordial germ cells in the recipient embryos.

[0054] After 2.5 days of incubation, a window approximately 2 cm in diameter was opened at the tip of the egg to expose the embryo. Approximately 1,000 to 5,000 drug-selected cells (suspended in 1 to 2 μl of PBS) were transplanted using a microglass needle into the blood of recipient embryos at Hamburger-Hamilton stages 13 to 15. The window was then sealed with cellophane tape, and the embryos were cultured at 38.5°C and 60 to 80% humidity until the chimeric individuals hatched (knock-in chimeric chicks (G0)). dIL-4 donor (conventional) knock-in male chimeras and dIL-4-OVA3UTR donor (improved) knock-in male chimeras were allowed to sexually mature and mated with wild-type females to obtain knock-in progeny (G1). The genotypes of the knock-in individuals were determined by PCR of the transgene-specific sequences using the genome from the G1 individual as a template. A dIL-4 donor (conventional) knock-in female (G1) and a dIL-4-OVA3UTR donor (improved) knock-in female (G1) were obtained, and these were sexually matured, and eggs were obtained from each.

[0055] Next, egg white from each egg and wild-type eggs (WT) was collected, and 2 μl was electrophoresed on a 5-20% acrylamide gel. The proteins contained in the egg white were visualized by Coomassie Brilliant Blue staining (Nacalai CBB Stain One). The results are shown in Figure 4. A clear band was observed at approximately 22 kDa that was present in both knock-in eggs but not in wild-type eggs, indicating that the foreign protein IL-4 had accumulated in the egg white. Furthermore, comparison of the CBB-stained band signals revealed that the amount of foreign protein IL-4 contained in the dIL-4-OVA3UTR donor (improved) knock-in eggs was significantly higher than that in the dIL-4 donor (conventional) knock-in eggs. Comparison of the expression levels of the foreign protein IL-4 (signal intensity of the foreign protein IL-4 by CBB staining) using the image analysis software NIH Image revealed that the expression level was significantly increased, two-fold, in the dIL-4-OVA3UTR donor (improved) knock-in eggs compared to the dIL-4 donor (conventional) knock-in eggs. The essential difference between the two is whether or not 650 bases of the 3'-untranslated region of chicken ovalbumin (SEQ ID NO: 9) were inserted into the 3'-end of the DNA encoding the foreign protein, and by inserting this base sequence, a remarkable effect of significantly increasing the expression level of the foreign protein of interest in an egg bioreactor was achieved for the first time.

[0056] Example 2: Analysis using poultry oviduct cells Experiments using quail-derived cells demonstrate that the remarkable effects obtained this time are not limited to chickens or the type of foreign gene expressed. The foreign genes in the conventional and improved donor vectors of Example 1 are replaced with secretory protein genes other than canine IL-4. The TEV sequence is also removed. These are then transfected into primary oviduct cells obtained from individual quails. In both experiments, the foreign gene is inserted into exon 2 of ovalbumin, and the foreign gene is expressed under the control of the ovalbumin promoter. The expression of the foreign gene product is examined using the same method as in Example 1. High expression is observed in cells into which the improved donor vector has been introduced.

[0057] Example 3 Analysis using poultry oviduct cells Experiments using poultry oviduct-derived cells demonstrated that the remarkable effects obtained this time are not limited to the type of foreign gene expressed. Figure 5 shows the foreign gene (human granulocyte-macrophage colony-stimulating factor (HMCF)) used in the experiment. Schematic diagrams of constructs (conventional and improved (constructs of the present invention)) containing ovotransferrin signal peptide and the subsequent four amino acids (amino acids 1-23 of the ovotransferrin immature protein, sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 29)) were placed on the 3' side of 2.8 kb (SEQ ID NO: 11) upstream of the ovalbumin translation initiation site. DNA (SEQ ID NO: 1) encoding the ovotransferrin signal peptide as a common signal peptide and the four amino acids following it (amino acids 1-23 of the ovotransferrin immature protein, sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 29)) was placed on the 3' side of this sequence. DNA (SEQ ID NO: 26) encoding the human GM-CSF mature protein was connected to the 3' side of this sequence as a foreign gene to be expressed. A termination codon (stop codon; TGA) was placed on the 3' side of this sequence, and a terminator sequence (the terminator sequence of BGH) for terminating transcription was placed on the 3' side of the termination codon. A sequence similar to that shown in FIG. 5 is provided. The construct is shown in the upper part of FIG. 5 as a conventional human GM-CSF expression construct for fallopian tube cells. The entire DNA sequence, i.e., 2.8 kb upstream of the ovalbumin translation initiation site, DNA encoding ovotransferrin signal peptide-human GM-CSF as the foreign gene to be expressed, and the subsequent terminator sequence, are shown in SEQ ID NO: 27. On the other hand, in the present invention, a construct was prepared in which 650 bases of the 3'-untranslated region of chicken ovalbumin (SEQ ID NO: 9) were placed on the 3' side of the foreign gene. The construct is shown in the lower part of FIG. 5 as an improved human GM-CSF expression construct, and the entire sequence is shown in SEQ ID NO: 28. The conventional and improved human GM-CSF expression constructs for fallopian tube cells were each expressed using the plasmid pBlue. The fragments were inserted into Script II (SK+) to give pBS-OVA-hGM-CSF (conventional type) and pBS-OVA-hGM-CSF (improved type).

[0058] Conventional and improved human GM-CSF oviduct cell expression constructs were expressed in primary cultured chicken oviduct cells, and the expression levels of the exogenous genes were compared. Primary cultured chicken oviduct cells were collected from 2-9 cm2 of oviduct tissue from 18-24 month-old White Leghorn females according to a previously reported technique (Muramatsu T. et al., Comp. Biochem. Physiol. Vol. 112B, pp. 209-216, 1995; Sanders M. M. and McKnight G. S., Endocrinology Vol. 116, pp. 398-405, 1985). Equal amounts of cells were seeded into 6-well cell culture plates at a confluency of 40-60%. Within 48 hours after collection, transfection was performed using 2 μg each of pBS-OVA-hGM-CSF (conventional) and pBS-OVA-hGM-CSF (improved) with 8 μl of FuGENE 6 (Promega). Three days after transfection, the medium was collected, and the foreign protein (human GM-CSF) contained in the medium was quantified using an ELISA kit (Human GM-CSF ELISA Kit, R&D Systems). The concentrations of the foreign gene product, human GM-CSF, were estimated to be 1.2 ng / ml and 2.8 ng / ml after transfection of the conventional and improved plasmids, respectively. This indicates that in poultry cells, particularly chicken oviduct cells, by inserting 650 bases of the 3' untranslated region of chicken ovalbumin into the 3' side of the foreign gene to be expressed, it is possible to significantly increase the amount of foreign gene product expressed and secreted by more than two-fold.

[0059] According to the present invention, it is possible to increase the expression of a foreign protein in avian cells, avian individuals, and avian eggs. The present invention can be used in the fields of medicine, food, livestock, etc. For example, the present invention can be used for protein production using a chicken egg bioreactor.

Claims

1. A method for increasing expression of a foreign protein, comprising expressing in poultry cells a construct comprising a gene encoding the foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is located 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

2. The method according to claim 1, wherein the base sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein has an identity of 85% or more to the base sequence shown in SEQ ID NO:

9.

3. The method according to claim 1, wherein the base sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein has an identity of 75% or more to the base sequence shown in SEQ ID NO:

25.

4. The method of any one of claims 1-3, wherein the poultry cells are oviduct-specific cells.

5. A method for producing poultry that expresses a foreign protein, comprising knocking in a construct comprising a gene encoding the foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein by placing it into an oviduct-specific locus, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is placed 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

6. The method according to claim 5, wherein the base sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein has an identity of 85% or more to the base sequence shown in SEQ ID NO:

9.

7. The method according to claim 5, wherein the base sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein has an identity of 75% or more to the base sequence shown in SEQ ID NO:

25.

8. The method of claim 5, wherein the fallopian tube-specific locus is the ovalbumin locus.

9. A poultry that has been knocked in by placing a construct comprising a gene encoding a foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein into an oviduct-specific locus, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is placed 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

10. The poultry according to claim 9, wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has an identity of 85% or more with the nucleotide sequence shown in SEQ ID NO:

9.

11. The poultry according to claim 9, wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the oviduct-specific protein has 75% or more identity with the nucleotide sequence shown in SEQ ID NO:

25.

12. The poultry of claim 9, wherein the oviduct-specific locus is the ovalbumin locus.

13. A construct comprising a gene encoding a foreign protein and the 3' untranslated region of a gene encoding an oviduct-specific protein, wherein the 3' untranslated region of the gene encoding the oviduct-specific protein is positioned 3' to the gene encoding the foreign protein, and the gene encoding the foreign protein contains a stop codon.

14. The construct according to claim 13, wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein comprises a nucleotide sequence having 85% or more identity to the nucleotide sequence shown in SEQ ID NO:

9.

15. The construct described in claim 13, wherein the nucleotide sequence of the 3' untranslated region of the gene encoding the fallopian tube-specific protein comprises a nucleotide sequence having 75% or more identity to the nucleotide sequence shown in SEQ ID NO:

25.

16. An expression vector comprising the construct of claim 13.

17. A poultry cell comprising the construct of claim 13 or the expression vector of claim 16.

18. A method for producing a foreign protein, comprising culturing the cell of claim 17.

19. Eggs laid by the poultry of any one of claims 9-12.

20. A method for producing a foreign protein, comprising obtaining the foreign protein from the egg of claim 19.

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