US20210269823A1

US20210269823A1 - Genetically-modified poultry egg

Info

Publication number: US20210269823A1
Application number: US17/070,276
Authority: US
Inventors: Isao Oishi; Kyoko YOSHII
Original assignee: National Institute of Advanced Industrial Science and Technology AIST
Current assignee: National Institute of Advanced Industrial Science and Technology AIST
Priority date: 2015-12-25
Filing date: 2020-10-14
Publication date: 2021-09-02
Also published as: JP6710825B2; US20190010515A1; US10858669B2; JPWO2017111144A1; JP2020078346A; JP6923232B2; WO2017111144A1

Abstract

Provided are a poultry knock-in egg and knock-out egg. The present invention pertains to a knock-out poultry egg in which at least one oviduct-specific gene has been knocked out, said gene being selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme, and at least one egg allergen protein has been reduced or eliminated, said protein being selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme.

Description

TECHNICAL FIELD

The present invention relates to an egg of knock-out poultry and an individual derived therefrom, and an egg and a thick albumen of knock-in poultry.
Further, the present invention relates to a method of preparing an expression product of an exogenous gene.

BACKGROUND

It has been attempted to induce the expression of human interferon β in an egg using a promoter containing a 2.8 kbp upstream region of a translation starting site of ovalbumin, or a promoter to which a further upstream estrogen-responsive enhancer element is attached to the 2.8 kbp region (Non-Patent Literature 1). In this example, a gene transfection is carried out using a lentiviral vector instead of using a knock-in method, so that a vector gene is inserted in a various location in the genome, and several vector genes are inserted. Consistent with such a gene transfection form, a concentration of human interferon β secreted in the egg significantly varies, and an average concentration in 6 chickens is 3.5 to 426 μg/ml. Further, data show that the concentration significantly varies among eggs derived from the same individual, indicating that the expression of interferon β is very unstable. Further, because the genes inserted in various locations of chromosomes are subjected to a gene silencing effect or the like, in general offspring (G2) of G1 chickens expressing interferon β at a relatively high level tend to reduce the expression level of interferon β.
In Non-Patent Literature 2, transgenic chimeric chickens (G0) expressing a Fv-Fc protein in the whole body are created using an actin promoter causing an expression in the whole body and a retroviral vector. It was confirmed that some G0 chickens express the Fv-Fc protein at a high concentration of 5 mg/ml. However, because the gene transfection is performed by viral infection in a chicken embryo, the gene transfection is achieved in a mosaic manner in which the presence/absence of gene insertion, the copy number of insertion, and a location of insertion differ between cells in the same individual. As a result, the G0 chimeric individual expressing the protein at a high concentration produces offsprings of transgenic individuals having the inserted genes varied in numbers and positions, making it difficult to completely transmit the character of the G0 chimeric individual to the offsprings. In fact, the expression level is reduced to 2 mg/ml or less in the G1 generation and 0.8 mg/ml or less in the G1 generation. Further, in many cases, an exogenous gene is not introduced in germline cells of the chimeric chicken infected by viruses. Thus, although one or several chickens that express the protein at a high level can be occasionally obtained in the G0 generation, it is difficult to propagate individuals having the same character and genetic information from such chickens. Such non-uniformity among G0 individuals or between G0 and G1 generations causes a fatal disadvantage for building so-called an “animal factory” in which a large number of chickens expressing an exogenous protein are grown to obtain a large number of eggs for mass production of the proteins.
Non-Patent Literature 4 shows an example in which an oviduct-specific gene, ovalbumin, is disrupted using a TALEN method. However, this literature only shows that a chick having a heterozygous deletion (+/−) in ovalbumin is obtained, and it is difficult to predict whether such poultry can produce an egg in the future, whether an egg having a null (−/−) genotype or an individual derived therefrom can be obtained, whether a homozygous knockout female (−/−) produces an egg, or whether an individual can hatch from an egg lacking the ovalbumin protein.

CITATION LIST

Non-Patent Literature

Non-Patent Literature 1: Lillico, S. G. et al. Oviduct-specific expression of two therapeutic proteins in transgenic hens. Proc Natl Acad Sci USA 104, 1771-1776 (2007)
Non-Patent Literature 2: Kamihira et al. High-Level Expression of Single-Chain Fv-Fc Fusion Protein in Serum and Egg White of Genetically Manipulated Chickens by Using a Retroviral Vector. Journal of Virology, p. 10864-10874 (2005)
Non-Patent Literature 3: van de Lavoir M C, Diamond J H, Leighton P A, Mather-Love C, Heyer B S, Bradshaw R, Kerchner A, Hooi L T, Gessaro T M, Swanberg S E et al: Germline transmission of genetically modified primordial germ cells. Nature 2006, 441(7094): 766-769
Non-Patent Literature 4: Park, T. S., Lee, H. J., Kim, K. H., Kim, J. S. & Han, J. Y. Targeted gene knockout in chickens mediated by TALENs. Proc Natl Acad Sci USA. 111, 12716-12721 (2014)

SUMMARY

Technical Problem

An object of the present invention is to provide a poultry egg in which an expression level of a protein encoded by an oviduct-specific gene is reduced or eliminated.
Further, another object of the present invention is to provide a poultry egg in which an exogenous gene is stably expressed, and a gene product thereof is highly expressed.
Further, another object of the present invention is to provide a technique for efficiently recovering an exogenous gene product, which is expressed in a chicken egg using a knock-in technique.

Solution to Problem

The present invention provides the following knock-out poultry egg and knock-in poultry egg. Further, the present invention provides a method of efficiently preparing an exogenous gene product from the knock-in poultry egg.
The present invention, in one aspect, relates to: [1] a knock-out poultry egg in which at least one oviduct-specific gene is knocked out, the gene being selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme, and at least one egg allergen protein is reduced or eliminated, the protein being selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme.
Further, in one embodiment of the present invention, [2] the knock-out poultry egg according to the item [1] above is characterized in that a base sequence encoding the knocked-out oviduct-specific gene includes deletion, substitution, or insertion of a base or bases in a region near a 5′ side or 3′ side of a PAM sequence.
Further, in one embodiment of the present invention, [3] the knock-out poultry egg according to the item [1] or [2] above is characterized in that the oviduct-specific gene is homozygously knocked-out and a genotype of the oviduct-specific gene is null (−/−).
Further, in one embodiment of the present invention, [4] the knock-out poultry egg according to any of the items [1] to [3] above is characterized in that the oviduct-specific gene is ovalbumin.
Further, in one embodiment of the present invention, [5] the knock-out poultry egg according to the item [4] above is characterized in that a base sequence encoding ovalbumin includes deletion, substitution, or insertion of a base or bases in a region corresponding to a base sequence represented by SEQ ID NO: 1 (OVATg1) and a vicinity thereof.
Further, in one embodiment of the present invention, [6] the knock-out poultry egg according to any of the items [1] to [3] above is characterized in that the oviduct-specific gene is an ovomucoid gene.
Further, in one embodiment of the present invention, [7] the knock-out poultry egg according to the item [6] above is characterized in that a base sequence encoding ovomucoid includes deletion, substitution, or insertion of a base or bases in a region corresponding to a base sequence represented by SEQ ID NO: 6 (OVMTg2) and a vicinity thereof.
Further, in one embodiment of the present invention, [8] the knock-out poultry egg according to the item [6] or [7] above is characterized by being substantially free of endogenous ovomucoid.
Further, the present invention, in another aspect, relates to:
[9] a knock-out poultry derived from the knock-out poultry egg according to any of the items [1] to [8] above.
Further, the present invention, in another aspect, relates to:
[10] a knock-in poultry egg, in which:
an exogenous gene under control of an oviduct-specific gene promoter is knocked-in as homozygous or heterozygous and an expression product of the exogenous gene is stably and highly expressed in an egg; and
the oviduct-specific gene promoter is at least one of promoters of oviduct-specific genes selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme.
Further, in one embodiment of the present invention, [11] the knock-in poultry egg according to the item [10] above is characterized in that the oviduct-specific gene promoter is an ovalbumin gene promoter, and the exogenous gene and a drug-resistant gene are both inserted in an exon 2 of an ovalbumin gene.
Further, in one embodiment of the present invention, [12] the knock-in poultry egg according to the item [10] or [11] above is characterized in that the exogenous gene is inserted in a region corresponding to a base sequence represented by SEQ ID NO: 1 (OVATg1) or a vicinity thereof or a region corresponding to a base sequence represented by SEQ ID NO: 24 (OVATg2) or a vicinity thereof in a base sequence encoding ovalbumin.
Further, in one embodiment of the present invention, [13] the knock-in poultry egg according to any of the items [10] to [12] above is characterized in that a protein encoded by the exogenous gene is contained in an amount of 1 mg or more per egg.
Further, in one embodiment of the present invention, [14] the knock-in poultry egg according to any of the items [10] to [13] above is characterized in that an expression product of the exogenous gene is dominantly expressed in a thick albumen.
Further, in one embodiment of the present invention, [15] the knock-in poultry egg according to any of the items [10] to [14] above is characterized in that the exogenous gene is a gene encoding interferon β, immunoglobulin, or collagen.
Further, in one embodiment of the present invention, [16] the knock-in poultry egg according to any of the items [10] to [15] above is characterized in that the exogenous gene is a gene derived from a human being.
Further, the present invention, in another aspect, relates to:
[17] a thick albumen derived from a knock-in poultry egg in which an exogenous gene under control of an ovalbumin gene promoter is knocked-in homozygously or heterozygously, the thick albumen dominantly containing an expression product of the exogenous gene that is stably and highly expressed.
Further, the present invention, in another aspect, relates to:
[18] a method of producing a knock-in poultry egg containing an expression product of an exogenous gene that is stably and highly expressed, the method comprising:
a step (a) of knocking-in the exogenous gene under control of an oviduct-specific gene promoter in a poultry primordial germ cell;
a step (b) of producing female poultry in which the exogenous gene under control of the oviduct-specific gene promoter is knocked-in as homozygous or heterozygous using the poultry germ cell; and
a step (c) obtaining a poultry egg expressing the exogenous gene from the female poultry.
Further, in one embodiment of the present invention, [19] the method of producing the knock-in poultry egg containing the expression product of the exogenous gene that is stably and highly expressed according to the item [18] above is characterized in that the step (a) is a step of introducing the exogenous gene by genome editing using (i) a donor construct that includes a 5′ side region of a translation starting site under control of the oviduct-specific gene promoter, the exogenous gene, a drug-resistant gene unit, and a 3′ side region of the translation starting site, and (ii) a vector that includes a target sequence and a different drug-resistant gene unit.
Further, in one embodiment of the present invention, [20] the method of producing the knock-in poultry egg containing the expression product of the exogenous gene that is stably and highly expressed according to the item [19] above is characterized in that:
the oviduct-specific gene promoter in the step (a) is an ovalbumin gene promoter; and
the step (d) is a step of recovering the expression product of the exogenous gene from a thick albumen of the poultry egg.
Further, in one embodiment of the present invention, [21] the method of producing the knock-in poultry egg containing the expression product of the exogenous gene that is stably and highly expressed according to the item [19] or [20] above is characterized in that the step (a) is a step of introducing the exogenous gene by CRISPR using (i) a donor construct that includes a 2.8 kb 5′ side region of a translation starting site of ovalbumin, the exogenous gene, a neomycin-resistant gene unit, and a 3.0 kb 3′ side region of the translation starting site of ovalbumin, and (ii) a vector that includes a base sequence represented by SEQ ID NO: 24 as the target sequence and a neomycin-resistant gene unit.
Further, the present invention, in another aspect, relates to:
[22] a method of preparing an expression product of an exogenous gene from a knock-in poultry egg, further comprising a step (d) of recovering the expression product of the exogenous gene from the poultry egg after the step (c) in the method according to any of items [18] to [21] above.

Effects of Invention

The knock-in poultry egg of the present invention is produced from a knock-in poultry individual in which an identical exogenous gene (a gene not derived from the poultry) is inserted in an identical location in the whole body, thus the difference in a protein expression level between individuals is small and genetic information and character of the exogenous gene can be properly transmitted over generations. Further, the expression of the exogenous gene can be restricted to an oviduct by performing gene knock-in at a location of the oviduct-specific gene. In this manner, a possibility of affecting a development process and the health of chicken is clearly lower as compared to a case where the exogenous gene is expressed in the whole body, which produces excellent effects. In addition, it is further preferable that the exogenous gene is expressed under control of a gene that is highly expressing in the albumen, such as ovomucoid, to increase an expression efficiency of the exogenous gene. Moreover, the knock-in chicken can be efficiently established by the gene knock-in using genome editing. It is confirmed that using such a new technique also allows the expression of the exogenous gene in the oviduct and the accumulation of the exogenous gene expression product in the albumen. Further, it is found, for the first time, that the exogenous gene-derived product mainly localizes to the thick albumen in the albumen. Based on this observation, the exogenous gene-derived product can be efficiently recovered by recovering a portion including the thick albumen from the egg containing the exogenous gene product, in a preferred embodiment, from the egg in which the exogenous gene is knocked-in at an albumen gene locus.
Because the oviduct-specific gene is knocked-out in the knock-out poultry egg of the present invention, the impact on the development is a matter of concern. However, it is confirmed by the inventor that such knock-out poultry can produce an egg.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a target sequence of a chicken ovalbumin gene (a target sequence of OVATg1). An sgRNA recognition site is indicated in capitals and a PAM sequence is indicated by an adjacent underline.

FIG. 2 shows a target sequence of a chicken ovomucoid gene (a target sequence of OVMTg2). An sgRNA recognition site is indicated in capitals and a PAM sequence is indicated by an adjacent underline.

FIG. 3 shows an example of disruption of the ovalbumin gene by CRISPR. An sgRNA recognition site is indicated in capitals (underlined) and a PAM sequence is indicated by an adjacent box. A deletion site of mutated sequence is indicated by a hyphen (-) and mutated sites are indicated in capitals. A translation starting site of OVATg1 is indicated by Met.

FIG. 4A shows an example of disruption of the ovomucoid gene by CRISPR. An sgRNA recognition site is indicated in capitals (underlined) and a PAM sequence is indicated by an adjacent box.

FIG. 4B (upper panel): An example of a chicken in which the ovomucoid gene is disrupted. A chicken (black) in a photograph is derived from a transplanted primordial germ cell and one allele of the ovomucoid gene has a 5-base deletion in a Tg2 region shown in FIG. 2. A base sequence of the Tg2 region in the chicken genome is analyzed both on a sense side and an antisense side. (lower panel): An example of ovomucoid gene mutations found in chicken individuals (F1 chickens). Deletions of 1 to 31 bases are found.

FIG. 5A shows knock-in of a human interferon β gene at an ovalbumin gene locus and demonstration of the knock-in by genome PCR. Primer 1 (P1) to primer 8 (P8) correspond to the following sequences. P1: SEQ ID NO: 15, P2: SEQ ID NO: 17, P3: SEQ ID NO: 16, P4: SEQ ID NO: 14, P5: SEQ ID NO: 18, P6: SEQ ID NO: 20, P7: SEQ ID NO: 21, P8: SEQ ID NO: 19. As a result of nested PCR, amplification products in predicted sizes are detected only in genome derived from the knock-in primordial germ cells (PGCs) (indicated by arrows in an image).

FIG. 5B shows knock-in of the human interferon β gene at the ovalbumin gene locus in a chimeric chicken sperm. Semen genomes from 4 chimeric chickens (411 to 414) and 1 negative control chicken (416, NC), and genome of knock-in cells (PCIFNKI #4) are amplified using primer sets of SEQ ID NO: 18 and 19 (3′ UTR), SEQ ID NO: 15 and 14 (5′ OVAp_out-IFN), and SEQ ID NO: 15 and 22 (5′ OVAp_out-OVA(ATG)). Detected bands in predicted sizes are indicated by “*”. In 411 and 412, knock-in signals having almost the same relative intensities as that of the positive control are detected.

FIG. 5C shows chickens in which the human interferon β gene is knocked-in at the ovalbumin gene locus. Photographs show chickens (female) which are offsprings of 411 and 412 in FIG. 5B. A PCR analysis of genomes derived from blood of the offsprings demonstrates that an IFN donor construct is knocked-in at the ovalbumin gene locus. A genome derived from blood of a wild-type (WT: a negative control) chicken, the genomes derived from blood of the knock-in chicken offsprings (KI), and a genome derived from a knock-in cell (KI PGC: a positive control) are amplified using primer sets of SEQ ID NO: 18 and 19 (a knock-in 3′ region), SEQ ID NO: 15 and 14 (a knock-in 5′ region), and SEQ ID NO: 15 and 22 (endogenous ovalbumin). Detected bands in predicted sizes are indicated by “*”. In the offsprings of 411 and 412, signals having the same patterns as that of the positive control are detected, indicating that the IFN donor construct is knocked-in at the ovalbumin gene locus in the chicken offsprings.

FIG. 6A shows target sequences (2 locations, the target sequences of OVATg1 and OVATg2) of the chicken ovalbumin gene. sgRNA recognition sites are indicated in capitals and PAM sequences are indicated by adjacent underlines.

FIG. 6B shows an efficiency of gene knock-in at the ovalbumin gene locus of the chicken primordial germ cell. The knock-in efficiency is the same between a transfection group 1 and a transfection group 2. Since the transfection group 2 has more cells, a transfection method of the transfection group 2 is more preferable. The knock-in efficiency seems to be higher in a transfection group 3 than that in the transfection group 2, thus a transfection method of the transfection group 3 is more preferable.

FIG. 7 shows knock-in of a human immunoglobulin gene at the ovalbumin gene locus and demonstration of the knock-in by genome PCR. Primer 1 (P1) to primer 8 (P8) correspond to the following sequences. P1: SEQ ID NO: 15, P2: SEQ ID NO: 17, P3: SEQ ID NO: 29, P4: SEQ ID NO: 28, P5: SEQ ID NO: 18, P6: SEQ ID NO: 20, P7: SEQ ID NO: 21, P8: SEQ ID NO: 19. As a result of nested PCR, amplification products in predicted sizes are detected only in the genome derived from the knock-in primordial germ cells (PGCs) (indicated by arrows in an image).

FIG. 8 shows knock-in of a human collagen gene at the ovalbumin gene locus and demonstration of the knock-in by genome PCR. Primer 1 (P1) to primer 8 (P8) correspond to the following sequences. P1: SEQ ID NO: 15, P2: SEQ ID NO: 17, P3: SEQ ID NO: 29, P4: SEQ ID NO: 28, P5: SEQ ID NO: 18, P6: SEQ ID NO: 20, P7: SEQ ID NO: 21, P8: SEQ ID NO: 31. As a result of nested PCR, amplification products in predicted sizes are detected only in the genome derived from the knock-in primordial germ cells (PGCs) (indicated by arrows in an image).

FIG. 9 shows an image of an egg produced from an interferon β knock-in female chicken. It is found that a thick albumen surrounding an egg yolk is cloudy.

FIG. 10 shows an image of western blotting of albumen components using an anti-human interferon β antibody. Recombinant human interferon β (indicated by an arrow) is expressed in an egg derived from a knock-in chicken. A thick albumen contains more human interferon β proteins than a thin albumen per unit volume. It is found that the thick albumen contains 100 times more human interferon β proteins than the thin albumen in terms of a relative concentration.

FIG. 11 shows a distribution of human interferon β in the albumen produced by the human interferon β knock-in chicken. In eggs derived from multiple chickens (KI egg 1 and 2), the thick albumen contains more interferon β proteins than the thin albumen (indicated by boxes). A concentration of recombinant human interferon β in the thick albumen is one tenth of the ovalbumin proteins present in a concentration of about 50 mg/ml (indicated by black arrows), thus the concentration of recombinant human interferon β is estimated to be about 5 mg/ml.

FIG. 12 shows how stably the interferon β protein is expressed in an egg produced from the interferon β knock-in chicken. Eggs were collected for a week (d1 to d7) and human interferon β contained in the thick albumen was identified by CBB staining. It is found that interferon β is stably expressed during the test period.

FIG. 13 shows an image of white precipitates obtained after centrifugation of the thick albumen. The thick albumen of the egg produced from the interferon β knock-in chicken is centrifuged without any treatment (1) or after various treatments (2 to 10) to compare amounts of white precipitate. The amounts of the white precipitates are reduced in 2 to 10 as compared to 1. In FIG. 13, a thick albumen liquid in an amount of 200 μl is added in each tube and subjected to the following treatments. The thick albumen liquid is added and mixed by inversion with 4 times volume (800 μl) of a 3M saturated arginine solution (tube 2), added with 4 times volume (800 μl) of the 3M saturated arginine solution and subjected to ultrasonic crushing (tube 3), added and mixed by inversion with a small amount of arginine (20 mg) and filled up with PBS to 1 ml (tube 4), added and mixed by inversion with a small amount of arginine hydrochloride (20 mg) and filled up with PBS to 1 ml (tube 5), filled up with PBS to 1 ml and subjected to the ultrasonic crushing (tube 6), added and mixed by inversion with arginine hydrochloride in a saturating amount or more (200 mg) (tube 7), added with twice volume (400 μl) of the 3M saturated arginine solution and subjected to the ultrasonic crushing (tube 8), added with a small amount of arginine hydrochloride (20 mg) and subjected to the ultrasonic crushing (tube 9), or added with a small amount of sodium chloride (40 mg) and subjected to the ultrasonic crushing (tube 10). Although the white precipitates were still observed after centrifugation at 20 k×g for 15 minutes, the amounts of the white precipitates were reduced by all of these treatments as compared to the case where no treatment was performed (tube 1). In particular, the amounts of the white precipitates were markedly reduced in the

tubes

3, 6, 8, and 9, which were subjected to the ultrasonic crushing.

FIG. 14 shows an image of electrophoresis of supernatants of the thick albumen after various treatments. The thick albumen without the centrifugation treatment (lane 0) and the supernatants of the tubes 1 to 10 in FIG. 13 (lanes 1 to 10) were subjected to electrophoresis after adjusting their loading amounts to be equal on the basis of the original amounts of the thick albumen. Bands of human interferon β are indicated by a black arrow.

FIG. 15 shows activity of human interferon β contained in an egg derived from the human interferon β knock-in chicken. The activity of human interferon β is detected in all of a thick albumen rough purification product, a centrifugation supernatant of the thick albumen, and the thin albumen.

FIG. 16 shows genomes of ovomucoid heterozygous knock-out (in a G1 generation: 5-base deletion in ORF) male and female, and genomes of ovomucoid heterozygous and homozygous knock-out and wild-type chickens obtained from the heterozygous knock-out chickens in the next generation.

FIG. 17 shows an image of electrophoresis of the thick albumen obtained from different G1 individuals. The thick albumen derived from a wild-type chicken (ctrl) and the thick albumen derived from 5 human interferon β knock-in chickens (#584, #766, #714, #645, and #640) are subjected to electrophoresis. Bands of human interferon β are indicated by an arrow.

FIG. 18 shows a comparison of activity between human interferon β contained in the egg derived from the human interferon β knock-in chicken (lower stage) and commercially available recombinant human interferon β (upper stage). In the image, culture supernatants of bioassay cells are added to a QUANTI-Blue substrate solution. The albumen supernatant and commercially available interferon β are serially diluted by 5-fold and added to the bioassay cells. Judging from a color change of the substrate solution, the albumen supernatant contains interferon β at a concentration of 625 or more times greater than a 0.01 μg/μl concentration of commercially available interferon β.

FIG. 19 shows the interferon β proteins (left panel) in the eggs derived from the human interferon β knock-in chickens in the G1 generation and G2 generation. Concentrations of human interferon β in the thick albumen of the eggs derived from G1 and G2 (3 female chickens) are approximately equal to each other. Further, the egg derived from G2 is cloudy as is the case for the egg derived from G1.

FIG. 20 shows an image of western blotting of the albumen derived from a chicken in which a human antibody gene is knocked-in at the ovalbumin gene locus using an anti-human immunoglobulin antibody. A recombinant human antibody (indicated by an arrow with a sign of hIgG) is expressed in an egg derived from the knock-in chicken (hIgG KI) but not in the albumen derived from a wild type (ctrl) (left panel). Further, the recombinant human antibody has the same electrophoretic mobility as a commercially available human antibody (Herceptin) under a non-reduced condition, indicating that the recombinant human antibody can form an antibody complex (right panel). A dilution ratio of the albumen is indicated by 1/2 k (1/2000), 1/200, and 1/20. Judging from band, intensities, a concentration of the antibody complex is 1 mg/ml or more.

FIG. 21 shows an egg derived from an ovomucoid homozygous knock-out (OVM−/− in a G2 generation). The egg is not markedly different from a wild-type egg in appearance (left panel) and the albumen and egg yolk of the egg are coagulated by heating (right panel), thus the egg can be processed similarly to the wild-type egg for cooking or other purposes.

FIG. 22 shows an ovomucoid homozygous knock-out individual in a G3 generation obtained by incubating an egg derived from the ovomucoid homozygous knock-out (OVM−/− in a G2 generation) (upper panel). The G3 generation was obtained by mating an OVM−/− female and OVM−/− male. A chicken can be developed without an endogenous ovomucoid gene or an ovomucoid gene product in the egg. It is found that the ovomucoid gene has a homozygous 5 bp deletion by a fragment analysis (lower panel).

FIG. 23 shows an image of eggs derived from 4 lines of interferon β knock-in female chickens. The thick albumen is cloudy in all 4 eggs as seen in FIG. 9.

DESCRIPTION OF EMBODIMENTS

In the present invention, a gene in a poultry primordial germ cell is modified by genome editing to obtain knock-in or knock-out female poultry that is derived from the genetically modified primordial germ cell, thereby obtaining a knock-in or knock-out poultry egg of the present invention from the knock-in or knock-out female.
In the present specification, the “knock-out poultry egg” includes eggs produced from female poultry having both heterozygous (+/−) and homozygous (−/−; null) genotypes of a knock-out gene. In a case where a knock-out gene is expressed in an oviduct and encodes an egg allergen accumulated in the albumen, an egg of the heterozygous knock-out poultry has a reduced amount of the egg allergen protein. On the other hand, an egg produced from the homozygous knock-out female poultry lacks the egg allergen protein.
In the present specification, the “knock-in poultry egg” includes eggs produced from female poultry having both heterozygous (+/−) genotype of, and fertilized eggs produced from poultry having homozygous (+/+) genotype of a knock-in gene. An egg produced from the female poultry having the homozygous (+/+) genotype of the knock-in gene contains more expression products of an exogenous gene than an egg produced from the female poultry having the heterozygous (+/−) genotype of the knock-in gene.
The genome editing is a technique for gene modification using a cleavage of double-stranded DNA and an error in repairing the cleavage and includes a nuclease capable of cleaving the target double-stranded DNA and a DNA recognition component that binds to or forms a complex with the nuclease. Examples of the genome editing technique include ZFN (zinc finger nuclease), TALEN, and CRISPR. For example, ZFN uses FokI (a nuclease) and a zinc finger motif (a DNA recognition component), TALEN uses FokI (a nuclease) and a TAL effector (a DNA recognition component), and CRISPR uses Cas9 (a nuclease) and a guide RNA (gRNA, a DNA recognition component). The nuclease used in the genome editing is only required to have nuclease activity, and a DNA polymerase, a recombinase, and the like may be used other than the nuclease.
Examples of the poultry include a chicken, a quail, a turkey, a duck, a goose, a long-tailed cock, a Japanese bantam, a pigeon, an ostrich, a green pheasant, a helmeted guineafowl, and the like. Preferable examples of the poultry include the chicken, the quail, and the like.
The primordial germ cell may be from male or female. The primordial germ cell of the poultry such as a chicken is a floating cell and cultured in the presence of a feeder cell, such as a BRL cell and STO cell. Alternatively, the primordial germ cell may be cultured in the absence of the feeder cell by adding an appropriate cytokine in a medium.
A gene modified by the genome editing is an oviduct-specific gene, and specific examples thereof include ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, lysozyme, and the like.
A gene function is eliminated by knock-out performed by the gene editing. In a case where at least one base is deleted or inserted in a gene by the gene editing, the gene function may be eliminated by a frame shift. The gene function may be eliminated without a frame shift by missing a part of amino acids. Further, the gene function may be eliminated by generating a stop codon by deletion or substitution.
In a case where an exogenous gene is knocked-in by the genome editing, the exogenous gene is preferably knocked-in at an oviduct-specific gene locus to obtain an egg containing an expression product of the exogenous gene instead of an expression product of the oviduct-specific gene. Examples of a protein as the expression product of the exogenous gene include various secreted proteins and peptides, and specific examples thereof include a functional peptide, such as an antibody (a monoclonal antibody) or a fragment thereof (e.g., scFv, Fab, Fab′, F(ab′)2, Fv, a single-chain antibody, scFv, dsFv, etc.), an enzyme, a hormone, a growth factor, a cytokine, an interferon, a collagen, an extracellular matrix molecule, and a vaccine, an agonistic protein, an antagonistic protein, and the like. In a case where the protein encoded by the exogenous gene is a biologically active protein that can be administered to human as a medicine, such a protein is derived from a mammal, preferably from human. Further, in a case where the protein encoded by the exogenous gene is an industrially applicable protein, such as a protein A and a protein constituting a spider thread, examples of the exogenous gene include a gene that encodes a protein derived from any organisms including a microorganism (bacteria, yeast, etc.), a plant, and an animal, or an artificial protein.
As the exogenous gene, a single gene or a plurality of genes may be used. In a case where a plurality of genes are used, the plurality of genes are expressed under control of the oviduct-specific gene. For example, the plurality of genes may be expressed by interposing a sequence such as IRES between the plurality of genes. Alternatively, the plurality of genes may be expressed by interposing a sequence encoding a 2A peptide or the like between the plurality of genes. In such a case, the plurality of genes are simultaneously expressed as a single peptide under control of an ovalbumin promoter and the peptide is cleaved to produce a plurality of proteins.
The exogenous protein may include an appropriate signal peptide. Codon usage of the exogenous protein may be changed to facilitate its expression in the poultry.
In a preferred embodiment of the knock-in poultry egg of the present invention, the expression product of the exogenous gene is dominantly expressed in the thick albumen. The term “dominant” herein refers to (a) a state in which an expression amount of the exogenous gene in the thick albumen is 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more by mass with respect to an expression amount of the exogenous gene in a whole knock-in egg or (b) a state in which an expression amount of the exogenous gene in the thick albumen is 1.1 times or more, preferably 2 times or more, more preferably 10 times or more of an expression amount of the exogenous gene in an egg other than the thick albumen in a relative concentration. The expression product of the knock-in exogenous gene is concentrated in the thick albumen and can thus be easily purified. Further, the expression product of the exogenous gene can be expressed in an active form. The thick albumen may become cloudy due to the expression product of the exogenous gene. However, a cloudy protein can be easily solubilized by an ultrasonic treatment, adding a solubilizing agent such as arginine hydrochloride, or the like.
In a preferred embodiment of the present invention, the expression product of the knock-in gene expressed in the thick albumen may be in a soluble form or in an insoluble form. The expression product of the knock-in gene in the insoluble form can be purified as an active protein. The expression product of the knock-in gene is preferably purified after solubilized. For purification, a conventional purification method, such as a column and dialysis, may be used. As the genome editing, a zinc finger, TALEN, and CRISPR can be mentioned. Of these, TALEN and CRISPR are preferable and CRISPR is more preferable. As a new genome editing method has been continuously developed, the present invention is not limited to the existing methods and any genome editing methods to be developed in the future may be used in the present invention.
When performing knock-in by the genome editing, it is preferable that a drug-resistant gene is stably integrated in the genome together with the useful exogenous gene to select a knock-in primordial germ cell by the drug-resistant gene. Examples of the drug-resistant gene include a neomycin-resistant gene (Neor), a hydromycin-resistant gene (Hygr), a puromycin-resistant gene (Puror), a blasticidin-resistant gene (blastr), a zeocin-resistant gene (Zeor), and the like. Of these, the neomycin-resistant gene (Neor) or the puromycin-resistant gene (Puror) is preferable.
When performing knock-in of the exogenous gene at the oviduct-specific gene locus, a translation starting site of the exogenous gene is preferably coincided with a translation starting site of the oviduct-specific gene. The exogenous gene may be introduced into the primordial germ cell as single- or double-stranded nucleic acids. The double-stranded nucleic acids may be introduced in a form of a plasmid vector, a BAC (bacterial artificial chromosome) vector, or the like. A gene sequence around the translation starting site of the oviduct-specific gene may be inserted immediately before the translation starting site of the exogenous gene to match the translation starting site of the exogenous gene with the translation starting site of the oviduct-specific gene by the knock-in.
In a case where the ovalbumin gene is selected as the oviduct-specific gene and the exogenous gene is introduced under control of the ovalbumin gene promoter, a 5′ end of the exogenous gene is preferably inserted in a base sequence encoding ovalbumin in a region corresponding to a base sequence represented by SEQ ID NO: 1 (OVATg1) or in a region corresponding to a base sequence represented by SEQ ID NO: 24 (OVATg2). More preferably, the translation starting site of the exogenous gene is inserted in the translation starting site of the ovalbumin gene.
In a case where the exogenous gene is knocked-in at the oviduct-specific gene locus to obtain a knock-in chicken individual and its egg, it is desirable that the albumen of the egg is recovered to recover the exogenous gene product. More desirably, a portion including the thick albumen surrounding the egg yolk is recovered to efficiently recover the exogenous gene product.
In a case where a gene function is eliminated by knock-out using the genome editing, the above-mentioned drug-resistant gene is preferably introduced into a primordial germ cell during the gene transfection by the gene editing to perform a selection on the basis of the drug-resistant gene. For the introduction of the drug-resistant gene and drug-based selection, the drug-resistant gene may be stably or transiently introduced. The drug-resistant gene is preferably transiently introduced in the case of the knock-out. Examples of the drug-resistant gene include the ones described above. Of these, the puromycin-resistant gene (Puror) or the zeocin-resistant gene (Zeor) is preferable. The drug-resistant gene may be introduced independently from a zinc finger, TALEN, or CRISPR plasmid or integrated into these plasmids. The drug-resistant gene is preferably integrated into the plasmid used for the genome editing.
In the present invention, performing knock-out of the oviduct-specific gene can induce deletion, substitution, or insertion of a base or bases in a base sequence encoding the oviduct-specific gene to be knocked-out and thus causes a frame shift or a nonsense mutation in the oviduct-specific gene, whereby a protein expression can be eliminated. For example, performing the knock-out by using CRISPR can induce deletion, substitution, or insertion of a base or bases in a region near a 5′ side or 3′ side of a PAM sequence. The region near the 5′ side or 3′ side of the PAM sequence is within, for example, about 1 to 50 bases, preferably about 1 to 15 bases, from the PAM sequence.
One embodiment of the present invention can include the knock-out poultry egg in which ovalbumin or ovomucoid is knocked-out as the oviduct-specific gene, although the present invention is not limited thereto. In a preferable embodiment of the knock-out of ovalbumin as the oviduct-specific gene, deletion, substitution, or insertion of a base or bases can be induced in a region corresponding to a base sequence represented by SEQ ID NO: 1 (OVATg1) and its vicinity. Further, in a preferable embodiment of the knock-out of ovomucoid as the oviduct-specific gene, deletion, substitution, or insertion of a base or bases can be induced in a region corresponding to a base sequence represented by SEQ ID NO: 6 (OVMTg2) and its vicinity.
In this description, for example, the “region corresponding to a base sequence represented by SEQ ID NO: 1 (OVATg1)” includes a corresponding region in a homolog of the ovomucoid gene, and a person skilled in the art can recognize the region corresponding to the base sequence in the poultry of interest.
The ovomucoid protein before secretion contains 210 amino acids (210aa) (an initiation methionine is counted as the first amino acid) and has a signal peptide from 1 to 24th aa. Further, the PAM sequence of SEQ ID NO: 1 (OVATg1) corresponds to 38 and 39th aa. Thus, in one embodiment of the present invention, the ovomucoid gene knock-out poultry egg expresses an ovomucoid mutant protein that lacks at least 160th aa and aa thereafter, preferably 100th aa and aa thereafter, more preferably 38th aa and aa thereafter.
Further, in a preferable embodiment of the present invention, the ovomucoid gene knock-out poultry egg is substantially free from endogenous ovomucoid. Being substantially free from endogenous ovomucoid means that endogenous ovomucoid is eliminated in an egg produced from an ovomucoid knock-out female poultry in which the ovomucoid gene is homozygously knocked-out.
Genetically modified poultry can be produced by a conventional method from the genetically modified poultry primordial germ cell that is obtained by the gene modification method in one embodiment of the present invention. Further, a (knock-in and knock-out) egg can be obtained from the genetically modified poultry. Specific procedures will be described below.
The genetically modified primordial germ cell is transplanted in a blastoderm, blood stream, or gonadal region of a recipient early embryo. Several hundreds to several thousands of the cells are transplanted by microinjection into the blood stream around the time of starting a blood circulation, preferably on the second or third day after the start of egg incubation. Further, the endogenous primordial germ cells of the recipient may be inactivated or reduced in number in advance by a drug or ionizing radiation before performing transplantation. The egg incubation is continued for the transplanted embryo according to a conventional method to obtain a transplanted individual. The transplantation and egg incubation may be performed in an ex-ovo culture system in which an eggshell is changed or a windowing method in which an eggshell is not changed. The hatched individual can be raised under a normal condition to sexual maturity to obtain a living individual (a chimeric individual). The chimeric individual is mated with a wild-type or genetically modified individual, or the genetically modified chimeric individual to produce a poultry offspring having the genetic modification derived from the transplanted cell. The primordial germ cell of the present invention obtained by the genome editing has high proliferation ability and differentiates into a large number of sperms or eggs having high fertilizability in the chimeric individual. In order to increase an efficiency of this process, a mating test may be performed after examining a frequency of gene modification in genomes of gametes or evaluating a contribution ratio of the transferred cells, or the offspring may be selected by a feather color. The genetically modified homozygous poultry can be obtained by mating the female chimeric poultry in which the genetically modified female primordial germ cells are transplanted with the male chimeric poultry in which the genetically modified male primordial germ cells are transplanted. Further, the present invention is not limited to internal fertilization of the poultry. In a case where a technique such as differentiating a primordial germ cell into a germ cell in vitro is developed in the future, the genetically modified poultry can be produced by artificial insemination or intracytoplasmic sperm injection using such a technique.
In FIG. 2, FIG. 4A, FIG. 4B, and FIG. 16, the PAM sequence of OVMTg2 is “agg”, however, NCBI databases include two kinds of sequences corresponding to chicken ovomucoid OVMTg2. Thus, the OVMTg2 sequence can be represented by TTTCCCAACGCTACAGACA(t or a)gg. The present invention includes all kinds of polymorphisms such as above.
In another embodiment of the present invention, the genome editing may be performed without culturing the primordial germ cell. In such a case, the endogenous primordial germ cell is genetically manipulated by infecting the early embryo with various viral vectors or injecting a plasmid vector as a liposome complex into a blood stream of the early embryo to establish a chimeric individual and a recombinant offspring. The primordial germ cell obtained by the genome editing includes the gene modification at a high frequency and has sufficiently high fertility to produce a recombinant poultry offspring or a genetically modified poultry offspring, and is thus useful in the present embodiment. In the present embodiment, the (endogenous) primordial germ cell can be genetically modified without culturing the primordial germ cell.
Examples of the viral vector used for gene manipulation by the genome editing include a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, and the like. These viral vectors may be used for the genome editing both in the cultured primordial germ cell and the endogenous primordial germ cell.
For example, in a case where the endogenous primordial germ cell is modified by the genome editing, the genome editing in the primordial germ cell may be performed by constructing a viral vector expressing a nuclease that recognizes and cleaves any target sequences and an sgRNA using a genome editing viral vector commercially available from various companies, processing the viral vector into an infectious form by packaging, and administering a resulting material into a place where the primordial germ cell exits, such as a blastoderm, blood stream, or gonadal region of the poultry early embryo. In this manner, a genetically modified individual and a genetically modified product can be obtained in the following generation. The commercially available genome editing viral vectors are offered by a number of companies worldwide, and examples thereof include an “AAVpro (registered trademark) CRISPR/Cas9 Helper Free System (AAV2)” available from Takara Bio Inc., which uses the adeno-associated viral vector, a “Lentiviral CRISPR/Cas9 System” available from System Biosciences, LLC, which uses the lentiviral vector, and the like. In a case where the gene modification involves knock-in, the viral vector required for the gene editing may be used with, for example, a viral vector, plasmid, Bac vector, or single- or double-stranded DNA including a gene to be knocked-in.
Further, a genome editing plasmid and a donor construct, either without or in combination with the viral vector, may be prepared in a cell membrane permeable form, such as a liposome complex, and administered into a place where the primordial germ cell exits, such as a blastoderm, blood stream, or gonadal region of the poultry early embryo, to perform the genome editing in the primordial germ cell and obtain a genetically modified individual and a genetically modified product in the following generation.
In the knock-in poultry egg of the present invention obtained by the above method, an expression product of the exogenous gene is stably and highly expressed in the egg. Herein, “the expression product of the exogenous gene being stably and highly expressed in the egg” means that a protein encoded by the exogenous gene is expressed in an amount of about 1 mg or more per egg in each egg derived from different individuals. The expression amount of the exogenous protein is preferably about 10 mg or more, more preferably 100 mg or more, per egg. Further, for example, in a case where the exogenous gene is knocked-in at the chicken oviduct-specific gene locus, the exogenous gene product (protein) is expressed in the thick albumen of the egg produced from the knock-in female chicken in a concentration of 5 mg/ml. This concentration is much higher than that obtained by a conventional gene transfection method that does not rely on knock-in and thus causes random gene insertions. Since the exogenous gene is inserted in an identical location, variation in expression level is small between individuals and in the same individual. Further, because the present invention uses a technique to perform knock-in at a translation starting site of a gene that is actually expressing in a chicken individual, the gene expression is not reduced by an effect of gene silencing or the like in a G2 generation or later.
In the case where the exogenous gene is knocked-in at the oviduct-specific gene locus by the present method, the exogenous gene product (protein) in the egg produced from the knock-in female chicken is distributed to the thick albumen at a higher concentration than that distributed to the thin albumen. Thus, the exogenous gene product can be efficiently recovered by recovering a portion including the thick albumen.
An egg deprived of an albumen allergen protein can be obtained by raising an albumen allergen gene homozygous knock-out chicken generated by the present method and obtaining an egg thereof. Such an egg is expected to have low allergenicity. Non-Patent Literature 4 discloses an example of a heterozygous ovalbumin knock-out chicken. However, it is impossible to predict whether a homozygous knock-out chicken can be obtained or whether such a homozygous knock-out chicken can produce an egg in light of the overall common technical knowledge at that time or from the literature.

EXAMPLES

Hereinafter, the present invention will be described in detail by way of examples.

Production Example 1

Genome Editing Using Chicken Male Primordial Germ Cell

Production Example 1-1

Gene Constructions for Knock-In at Ovalbumin (OVA) Gene Locus and Knock-Out of Ovomucoid (OVM)

A CRISPR method was applied to a chicken male primordial germ cell line for targeting an ovalbumin and ovomucoid genes. As shown in FIG. 1 (ovalbumin) and FIG. 2 (ovomucoid), OVATg1 and OVMTg2 were tested respectively if they were suitable as targets.
A CRISPR plasmid for targeting the target sequence of the ovalbumin gene shown in FIG. 1 was constructed.
First, for targeting SEQ ID NO: 1 (OVATg1), oligo DNAs represented by SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized, subjected to 5′ phosphorylation by T4 polynucleotide kinase, and then annealed by heating a mixture of both oligo DNAs to 98° C. and slowly cooling the mixture to the room temperature. This DNA fragment was inserted into a BbsI cleavage site of a plasmid px330-Puro^rin which a puromycin-resistant gene unit represented by SEQ ID NO: 4 was inserted into a NotI site of a plasmid px330 (AddGENE, USA) to obtain px330-Puro^r-OVATg1. Further, the puromycin-resistant gene unit in px330-Puro^r-OVATg1 was replaced with a neomycin-resistant gene unit represented by SEQ ID NO: 5 to construct px330-Neo^r-OVATg1.
A CRISPR plasmid for targeting the target sequence of the ovomucoid gene shown in FIG. 2 was constructed. For targeting SEQ ID NO: 6 (OVMTg2), oligo DNAs represented by SEQ ID NO: 7 and SEQ ID NO: 8 were synthesized, phosphorylated, annealed, and inserted into a BbsI cleavage site of the plasmid px330-Puro^rto obtain px330-Puro^r-OVMTg2.

Production Example 1-2

Knock-Out of Ovalbumin and Ovomucoid Genes

A chicken male primordial germ cell was collected from the blood stream of a male embryo of Barred Plymouth Rock to establish a cell line (the cell line was prepared according to Non-Patent Literature 3). The cell line was transiently transfected with the above genes (plasmids). After 1×10⁵to 5×10⁵male primordial germ line cells were rinsed with PBS and suspended into OPTI-MEM, 1.6 μg of px330-Neo^r-OVATg1 was transfected into the cells using 3 μl of Lipofectamine 2000 (Life Technologies, USA). Specifically, Lipofectamine 2000 and the plasmid were mixed in the 80 μl of OPTI-MEM, and the resulting mixture was added to the male primordial germ line cells. The primordial germ line cells mixed with the mixture were left still for about 5 minutes at the room temperature and then added with 500 μl of medium containing no antibiotics. After being left still at 37° C. for about 1 to 4 hours, the primordial germ line cells were seeded onto feeder cells. The cell culture was added with neomycin (G418 disulfate salt, Nacalai, Japan) at a final concentration of 0.5 mg/ml from day 2 to day 4 after gene transfection. Then, the primordial germ line cells were rinsed to remove neomycin and cultured for another 1 to 2 weeks. After the cultured cells were collected and their genomic DNA was extracted, a PCR method was performed to amplify a part of the ovalbumin gene with oligo DNA primers represented by SEQ ID NO: 9 and SEQ ID NO: 10. The amplified DNA was sub-cloned into a TA vector (pGEM-T Easy, Promega, USA) to analyze a genome base sequence of a region including SEQ ID NO: 1 (OVATg1). As shown in FIG. 3, mutations including a deletion or substitution of the start codon were confirmed.
Next, a similar analysis was performed by targeting the ovomucoid gene. As described above, 1×10⁵to 5×10⁵male primordial germ line cells were transfected with 1.6 μg of px330-Puro^r-OVMTg2 using Lipofectamine 2000. The cell culture was added with puromycin at a final concentration of 1 μg/ml from day 2 to day 4 after gene transfection. The cells were rinsed to remove puromycin and cultured for another 1 to 2 weeks. After the cells were collected and their genomic DNA was extracted, a PCR method was performed to amplify a part of the ovomucoid gene with oligo DNA primers represented by SEQ ID NO: 11 and SEQ ID NO: 12. The amplified DNA was sub-cloned into a TA vector to analyze a genome base sequence of a region including SEQ ID NO: 6 (OVMTg2). Gene deletion was observed in 21 out of 23 clones analyzed (91%) in the region including SEQ ID NO: 6 (OVMTg2) of the ovomucoid gene. On the other hand, gene deletion was observed in 0 out of 24 clones (0%) in a control group not selected by a drug. An example of gene mutations found in the region including OVMTg2 are shown in FIG. 4A. These results show that a mutation efficiency can be markedly increased in the genome editing of a gene of the poultry primordial germ cell by introducing a drug-resistant gene and transiently performing a drug selection, in particular, a high mutation efficiency can be obtained by the puromycin-resistant gene and the drug selection using puromycin.

Production Example 1-3

Establishing Genome Edited Chicken

The px330-Puro^r-OVMTg2 plasmid was transfected into Barred Plymouth Rock primordial germ cells in a manner described in Production example 1-2 and the drug selected cells were cultured. The cultured cells were transplanted into a blood stream of a 2.5-day-old White Leghorn embryo (a recipient embryo) by microinjection. Prior to transplantation, a fertilized egg was irradiated with ionizing radiation at 5 Gy or 6 Gy before incubation to reduce the number of endogenous primordial germ cells in the recipient embryo. The ionizing radiation was performed by gamma irradiation using a Gammacell 40 irradiator (Atomic Energy of Canada Ltd.).
After 2.5 days of incubation, a window having a diameter of about 2 cm was cut in the egg shell on a protruding side to expose an embryo. About 1,000 to 5,000 drug selected cells (suspended in 1 to 2 μl PBS) were transplanted in the blood stream of the recipient embryo at a hamburger-hamilton stage of 13 to 15 using a glass micropipette. After the window was sealed by a cellophane tape, the egg was incubated and hatched at a temperature of 38.5° C. and a humidity of 60 to 80% (a chimeric chick (G0)). Eight male chimeric chicks were raised to sexual maturity and their sperms were collected. After the genomic DNA was extracted from the sperms, a PCR method was performed to amplify a part of the ovomucoid gene with oligo DNA primers represented by SEQ ID NO: 11 and SEQ ID NO: 12. The amplified DNA was sub-cloned into a TA vector to analyze a genome base sequence of the region including SEQ ID NO: 6 (OVMTg2). Chimeric chickens #372 and #376 having a high mutation frequency (in both cases, the ovomucoid gene was mutated in 10 out of 11 clones after sub-cloning) were mated with wild-type Barred Plymouth Rock females to find 11 and 6 ovomucoid mutated chickens (chicks) out of 19 and 14 offsprings, respectively. One example of mutation in the ovomucoid gene is shown in an upper panel of FIG. 4B. This individual has a 5-base deletion immediately after a signal peptide of the ovomucoid protein, which causes a frame shift mutation in one allele of the ovomucoid gene. Further, representative examples of mutations (gene deletions) found around the target region of ovomucoid genome are shown in a lower panel of FIG. 4B. Several female and male individuals of ovomucoid heterozygous knock-out having frame shift mutations immediately after the signal peptide of the ovomucoid protein as represented above were obtained, allowing the production of a homozygous ovomucoid knock-out chicken by mating these individuals after sexual maturity.

Production Example 2

Human Interferon Gene Knock-In at Ovalbumin Gene Locus

Production Example 2-1

Knock-In for Establishing Primordial Germ Cell and Establishment of Knock-In Chimeric Chicken

In order to insert an exogenous gene (human interferon β; IFNβ) at a translation starting site of the ovalbumin gene, a donor construct (an IFNβ donor construct) including a human interferon β gene represented by SEQ ID NO: 13 was created. This donor construct contains an about 2.8 kb 5′ side of a translation starting site of ovalbumin, the human interferon β gene, a drug-resistant gene unit (PGK-puro^r), and an about 3.0 kb 3′ side of the translation starting site of ovalbumin. This donor construct was inserted in a plasmid pBlue ScriptII (SK+) (Stratagene, USA, current Agilent Technologies) to create a pBS-IFNβ donor. As with production example 1, 1×10⁵to 5×10⁵primordial germ line cells were simultaneously transfected with 0.8 μg of px330-Neo^r-OVATg1 and 0.8 μg of the pBS-IFNβ donor using Lipofectamine 2000. The cell culture was added with puromycin at a final concentration of 1 μg/ml on the third day after gene transfection. After the medium was replaced as needed, cells capable of growing in the presence of puromycin at a final concentration of 1 μg/ml were recovered to prepare their genomic DNA. The genome PCR was performed to confirm that the donor construct was knocked-in at the ovalbumin gene locus. PCR in the 5′ region was conducted as described below using a primer recognizing the exogenous gene on the donor construct and a primer recognizing a 5′ region of ovalbumin not included in the donor construct. The PCR was conducted using an antisense primer recognizing interferon β, represented by SEQ ID NO: 14, and a sense primer recognizing a region of an about 3.0 kb 5′ side of the translation starting site of ovalbumin, represented by SEQ ID NO: 15. Another round of PCR (nested PCR) was conducted with an amplification product using an antisense primer recognizing interferon β, represented by SEQ ID NO: 16, and a sense primer recognizing an about 2.85 kb 5′ side of the translation starting site of ovalbumin not included in the donor construct, represented by SEQ ID NO: 17. As shown in FIG. 5A, when px330-Neo^r-OVATg1 and the donor construct were transfected and genome derived from the drug selected primordial germ cells (knock-in PGCs) was used as a template, an amplification product was detected at a position of about 2.9 k that was expected when the donor construct was inserted. In contrast, when genome derived from the control primordial germ cells not subjected to gene transfection (control PGCs) was used as a template, such an amplification product was not detected.
Similarly, a 3′ region was examined by the genome PCR using a primer recognizing the exogenous gene on the donor construct and a primer recognizing a 3′ region of ovalbumin not included in the donor construct. The PCR was conducted using a sense primer recognizing the drug-resistant gene unit, represented by SEQ ID NO: 18, and an antisense primer recognizing a region of an about 3.4 kb 3′ side of the translation starting site of ovalbumin, represented by SEQ ID NO: 19. Another round of PCR (nested PCR) was conducted with an amplification product using a sense primer recognizing the drug-resistant gene unit, represented by SEQ ID NO: 20, and a sense primer recognizing an about 3.2 kb 3′ side of the translation starting site of ovalbumin not included in the donor construct, represented by SEQ ID NO: 21. As shown in FIG. 5A, when px330-Neo^r-OVATg1 and the donor construct were transfected and genome derived from the drug selected primordial germ cells (knock-in PGCs) was used as a template, an amplification product was detected at a position of about 3.4 k that was expected when the donor construct was inserted. In contrast, when genome derived from the control primordial germ cells not subjected to gene transfection (control PGCs) was used as a template, such an amplification product was not detected. These results suggest that the drug selected cell group includes a cell in which the donor construct including the exogenous gene portion is knocked-in at the ovalbumin gene locus.
The primordial germ cells containing the cell in which the IFNβ donor construct was knocked-in were transplanted into recipient embryos by the same method as described in Production example 1-3 and the embryos were incubated to obtain 4 chimeric male chickens (#411 to #414). After semen was collected from each chicken to isolate genomic DNA, PCR was conducted using primers represented by SEQ ID NO: 18 and SEQ ID NO: 19 (for amplifying a 3′ side of interferon knocked-in at the ovalbumin gene), primers represented by SEQ ID NO: 15 and SEQ ID NO: 14 (for amplifying a 5′ side of interferon knocked-in at the ovalbumin gene), and primers represented by SEQ ID NO: 15 and SEQ ID NO: 22 (for amplifying ovalbumin without a knock-in event) (FIG. 5B). The chimeric chickens #411 and #412 show signals that clearly prove the interferon knock-in at the ovalbumin gene locus at both the 3′ side and 5′ side. In particular, signal intensities of #411 are comparable to that of the transplanted parental cell line, suggesting that the sperms contain the interferon knock-in cells to the same extent as the parental cell line.
The chimeric chickens #411 and #412 were mated with wild-type female chickens (Barred Plymouth Rock) to obtain 28 and 19 offsprings, respectively. After wing shafts were collected from the offsprings to isolate their genomic DNA, PCR was conducted as described above using primers represented by SEQ ID NO: 18 and SEQ ID NO: 19 (for amplifying the 3′ side of interferon knocked-in at the ovalbumin gene), primers represented by SEQ ID NO: 15 and SEQ ID NO: 14 (for amplifying the 5′ side of interferon knocked-in at the ovalbumin gene), and primers represented by SEQ ID NO: 15 and SEQ ID NO: 22 (for amplifying ovalbumin without a knock-in event). Further, the same PCR was conducted with genome derived from a wild-type wing shaft (a negative control (NC)) and genome derived from the transplanted interferon donor vector knock-in primordial germ cells (a positive control (PC)). Eight out of 28 offsprings derived from #411 and 5 out of 19 offsprings derived from #412 showed signals that clearly proved the interferon knock-in at the ovalbumin gene locus at both the 3′ side and 5′ side, as were seen in the positive control. FIG. 5C shows an image of electrophoresis of PCR products from the offspring (female) derived from #411 and the offspring (female) derived from #412. These results indicate that the interferon donor vector is knocked-in at the ovalbumin gene locus in these female chicken offsprings.

Production Example 2-2

Improvement of Knock-In Efficiency

Studies were conducted to improve a gene knock-in efficiency. First, the drug resistance unit of the interferon β donor construct in the above production example 2-1 was changed from PGK-Puro^rto SV40Pe-Neo^r(SEQ ID NO: 23) to create an IFNβ-Neo donor construct <SEQ ID NO: 33>. This donor construct contains an about 2.8 kb 5′ side of the translation starting site of ovalbumin, the human interferon β gene, the drug resistant gene unit (SV40Pe-Neo^r), and an about 3.0 kb 3′ side of the translation starting site of ovalbumin. This donor construct was inserted in the plasmid pBlue ScriptII (SK+) to create a pBS-IFNβ-Neo donor. Further, as shown in FIG. 6A, a CRISPR plasmid for targeting the target sequence OVATg2 (SEQ ID NO: 24) of ovalbumin partially overlapping with OVATg1 was constructed. Oligo DNAs represented by SEQ ID NO: 25 and SEQ ID NO: 26 were synthesized and, as described in production example 1-1, they were phosphorylated, annealed, and inserted, as a DNA fragment, into the BbsI cleavage site of px330 to construct a plasmid, px330-Puro^r-OVATg2, which also has the puromycin-resistant unit represented by SEQ ID NO: 4 inserted into a NotI site. After about 5×10⁵primordial germ line cells were prepared and divided into 3 groups, as described in production example 2-1, the cells were simultaneously transfected with 0.8 μg of px330-Neo^r-OVATg1 and 0.8 μg of the pBS-IFN donor (including the puromycin-resistant gene unit) (transfection group 1), or 0.8 μg of px330-Puro^r-OVATg1 and 0.8 μg of the pBS-IFNβ-Neo donor (transfection group 2), or 0.8 μg of px330-Puro^r-OVATg2 and 0.8 μg of the pBS-IFNβ-Neo donor (transfection group 3) using Lipofectamine 2000. The transfected group 1 was added with puromycin at a final concentration of 1 μg/ml on the third day after gene transfection as was the case in production example 2-1. On the other hand, the transfection group 2 and transfection group 3 were cultured in the presence of puromycin at a final concentration of 1 μg/ml from day 2 to day 4 after gene transfection as was the case in production example 1-2. After rinsed, the cells were added with neomycin at a final concentration of 0.5 mg/ml and cultured. The number of cells was counted in each transfection group on day 24 after gene transfection. As a result, the transfection group 1 had 2×10⁴drug-resistant cells while the transfection groups 2 and 3 had 1×10⁵drug-resistant cells. Further, the cells in each transfection group were recovered to prepare their genomic DNA. Then, as described in production example 2-1, PCR was conducted using primers represented by SEQ ID NO: 18 and SEQ ID NO: 19 (for amplifying a 3′ side of interferon knocked-in at the ovalbumin gene), primers represented by SEQ ID NO: 15 and SEQ ID NO: 14 (for amplifying a 3′ side of interferon knocked-in at the ovalbumin gene), and primers represented by SEQ ID NO: 15 and SEQ ID NO: 22 (for amplifying ovalbumin without a knock-in event). Note that a primer represented by SEQ ID NO: 71 was used instead of the primer represented by SEQ ID NO: 18 in the transfection groups 2 and 3 (FIG. 6B). There is no considerable difference between the transfection groups 1 and 2 in terms of a PCR signal intensity ratio, suggesting that preparation of desired cells can be quicker in the method of the transfection group 2, in which the cells are briefly selected by puromycin and then selected by neomycin. Further, as compared to the transfection group 2, the transfection group 3 rarely contains ovalbumin not having a knock-in event, suggesting a better knock-in efficiency in the transfection group 3. These results suggest that the exogenous gene can be quickly and highly efficiently knocked-in at the ovalbumin gene locus by transfecting the primordial germ cells with the CRISPR construct targeting the OVATg2 sequence, inserting the puromycin-resistant gene and the neomycin-resistant gene into the CRISPR construct and the donor construct, respectively, and temporarily selecting with puromycin, and then selecting with neomycin.

Production Example 3

Human Antibody Gene Knock-In at Ovalbumin Gene Locus

Human interferon β in the interferon β donor construct in the above production example 2-1 was replaced with a human immunoglobulin gene represented by SEQ ID NO: 27 to create a donor construct (an immunoglobulin donor construct). In this donor construct, genes encoding an albumen lysozyme signal peptide, a human immunoglobulin heavy chain, a cleavage target sequence of furin protein, a 2A self-processing peptide, an albumen lysozyme signal peptide, and a human immunoglobulin light chain gene are arranged in tandem at a downstream of an about 2.8 kb 5′ side of the translation starting site of ovalbumin, which are followed by the drug-resistant gene unit (PGK-Puro^r) and an about 3.0 kb 3′ side of the translation starting site of ovalbumin. This donor construct is transcribed and translated to express an antibody protein composed of immunoglobulin heavy chains and light chains.
The immunoglobulin donor construct was inserted into the plasmid pBlue ScriptII (SK+) to create a pBS-immunoglobulin donor (a pBS-IgG(Hc+Lc) donor). After the donor construct was knocked-in into the male chicken primordial germ cells by the same method used for the pBS-IFNβ donor described above, the primordial germ cells were selected by puromycin and PCR was performed using genome of the selected cells as a template. The PCR in a 5′ side was conducted using a primer represented by SEQ ID NO: 15 and an antisense primer recognizing the albumen lysozyme signal peptide, represented by SEQ ID NO: 28. Another round of PCR (nested PCR) was conducted with an amplification product using a primer represented by SEQ ID NO: 17 and an antisense primer recognizing the albumen lysozyme signal peptide, represented by SEQ ID NO: 29. The PCR in a 3′ side was conducted in the same manner as for the knock-in of the EGFP donor and pBS-IFNβ donor described above. That is, the PCR was conducted using the primers represented by SEQ ID NO: 18 and SEQ ID NO: 19 and nested PCR was conducted with an amplification product using the primers represented by SEQ ID NO: 20 and SEQ ID NO: 21. As shown in FIG. 7, knock-in at the ovalbumin gene in the primordial germ cells was also observed using the immunoglobulin donor.
Further, similarly to production example 2-2, the drug resistance unit of the immunoglobulin donor construct was changed from PGK-Puro^rto SV40Pe-Neo^r(SEQ ID NO: 23) to create an immunoglobulin-Neo donor construct (SEQ ID NO: 30). In this donor construct, genes encoding an albumen lysozyme signal peptide, a human immunoglobulin heavy chain, a cleavage target sequence of furin protein, a 2A self-processing peptide, an albumen lysozyme signal peptide, and a human immunoglobulin light chain gene are arranged in tandem at a downstream of an about 2.8 kb 5′ side of the translation starting site of ovalbumin, which are followed by the drug-resistant gene unit (SV40Pe-Neo^r) and an about 3.0 kb 3′ side of the translation starting site of ovalbumin. This donor construct was inserted into the plasmid pBlue ScriptII (SK+) to create a pBS-immunoglobulin-Neo donor. With the same method used in production example 1-2, about 2×10⁵primordial germ line cells were transfected with 0.8 μg of px330-Puro^r-OVATg2 and 0.8 μg of the pBS-immunoglobulin-Neo donor using 3 μl of Lipofectamine 2000. The cells were cultured in the presence of puromycin at a final concentration of 1 μg/ml from day 2 to day 4 after gene transfection. After rinsed, the cells were added with neomycin at a final concentration of 0.5 mg/ml and cultured. A cell group containing immunoglobulin knock-in cells was obtained after about 3 weeks of culturing. Using the same method as described in production example 1-3, the cell group was transplanted into a recipient embryo and the embryo was incubated to establish an immunoglobulin knock-in germline chimeric chicken. A chicken in which the human immunoglobulin gene is knocked-in at the ovalbumin gene locus is obtained in the following generation and such a chicken expresses an antibody protein composed of the human immunoglobulin heavy chains and light chains in the albumen.

Production Example 4

Human Collagen Gene Knock-In at Ovalbumin Gene Locus
Human interferon β in the interferon β donor construct in the above production example 2-1 was replaced with a human type I collagen gene represented by SEQ ID NO: 31 to create a donor construct (a collagen donor construct). In this donor construct, genes encoding an albumen lysozyme signal peptide, a human type I collagen α1 chain (COLLAGEN1A1), a cleavage target sequence of a furin protein, a 2A self-processing peptide, an albumen lysozyme signal peptide, and a human type I collagen α2 chain (COLLAGEN1A2) gene are arranged in tandem at a downstream of an about 2.8 kb 5′ side of the translation starting site of ovalbumin, which are followed by the drug-resistant gene unit (PGK-Puro^r) and an about 3.0 kb 3′ side of the translation starting site of ovalbumin. This donor construct is transcribed and translated to express a type I collagen protein composed of the human type I collagen α1 chains and α2 chain.
The collagen donor construct was inserted into the plasmid pBlue ScriptII (SK+) to create a pBS-COL1(A1+A2) donor. The pBS-COL1(A1+A2) donor was knocked-in into the male chicken primordial germ cells by the same method used for the ppBS-IFNβ donor and pBS-IgG(Hc+Lc) donor described above. After knock-in, the primordial germ cells were selected by puromycin and PCR was conducted using genome of the selected cells as a template. The PCR in a 5′ side was conducted in the same manner as for the pBS-IgG(Hc+Lc) donor. That is, the PCR was conducted using the primer represented by SEQ ID NO: 15 and the antisense primer recognizing the albumen lysozyme signal peptide, represented by SEQ ID NO: 28. Then, another round of PCR (nested PCR) was conducted with an amplification product using the primer represented by SEQ ID NO: 17 and the antisense primer recognizing the albumen lysozyme signal peptide, represented by SEQ ID NO: 29. The PCR in a 3′ side was conducted in the same manner as for the knock-in of the pBS-IFNβ donor and pBS-IgG(Hc+Lc) donor described above. That is, the PCR was conducted using the primers represented by SEQ ID NO: 18 and SEQ ID NO: 19 and then nested PCR was conducted with an amplification product using the primers represented by SEQ ID NO: 20 and SEQ ID NO: 21. As shown in FIG. 8, knock-in at the ovalbumin gene in the primordial germ cells was also observed using the collagen donor.
Further, similarly to production example 2-2, the drug resistance unit of the collagen donor construct was changed from PGK-Puro^rto SV40Pe-Neo^r(SEQ ID NO: 23) to create a collagen-Neo donor construct (SEQ ID NO: 32). In this donor construct, genes encoding an albumen lysozyme signal peptide, the human type I collagen α1 chain (COLLAGEN1A1), the cleavage target sequence of furin protein, the 2A self-processing peptide, the albumen lysozyme signal peptide, and the human type I collagen α2 chain (COLLAGEN1A2) gene are arranged in tandem at a downstream of an about 2.8 kb 5′ side of the translation starting site of ovalbumin, which are followed by the drug-resistant gene unit (SV40Pe-Neo^r) and an about 3.0 kb 3′ side of the translation starting site of ovalbumin. This donor construct was inserted into the plasmid pBlue ScriptII (SK+) to create a pBS-collagen-Neo donor. Using the same method as described in production example 1-2, about 2×10⁵primordial germ line cells were transfected with 0.8 μg of px330-Puro^r-OVATg2 and 0.8 μg of the pBS-collagen-Neo donor using 3 μl of Lipofectamine 2000. The cells were cultured in the presence of puromycin at a final concentration of 1 μg/ml from day 2 to day 4 after gene transfection. After rinsed, the cells were added with neomycin at a final concentration of 0.5 mg/ml and cultured. A cell group containing collagen knock-in cells was obtained after about 3 weeks of culturing. Using the same method as described in production example 1-3, the cell group was transplanted into a recipient embryo and the embryo was incubated to establish an immunoglobulin knock-in germline chimeric chicken. A chicken in which the human immunoglobulin gene is knocked-in at the ovalbumin gene locus is obtained in the following generation and such a chicken expresses a protein complex composed of the human type I collagen α1 and α2 in the albumen.

Example 1

As described in above production example 2-1, the female and male chickens in which the human interferon β donor vector was knocked-in at the translation starting site of the ovalbumin gene locus were established.

(1) Characteristics of Knock-In Chicken and Knock-In Egg

The established knock-in chickens reached sexual maturity without showing a developmental abnormality or significant disease condition. The female knock-in chicken laid eggs. A content of the egg was examined by opening an eggshell. As a result, a cloudy thick albumen was found around an egg yolk. On the other hand, similar to a wild-type egg, a thin albumen having a low viscosity was observed. A typical image of opened egg is shown in FIG. 9.

(2) Identification of Interferon and Possible Enrichment in Thick Albumen

Next, the presence of human interferon β in the albumen was examined. The thick albumen and the thin albumen were recovered by a dropper and added with an equal volume of a sample buffer (0.125M Tris pH6.8, 10% 2-ME, 4% SDS, 10% glycerol, 0.1% BPB). These samples were serially diluted 10 folds 3 times, separated by electrophoresis using a 5-20% acrylamide gel, and transferred to a PVDF membrane. After the membrane was blocked from a non-specific binding of an antibody molecule by skim milk, the membrane was subjected to western blotting using an anti-human interferon β antibody (abcam ab85803, a rabbit polyclonal antibody) diluted 1,000 times as a primary antibody and an anti-rabbit HRP conjugated antibody (GE Healthcare NA934V) diluted 1,000 times as a secondary antibody. A result is shown in FIG. 10.
The antibodies detected bands of about 30 kDa at the same position as that of purified recombinant human interferon β (WAKO rhIFN-β). Further, this band was not detected in a wild-type egg at all. In FIG. 10, numbers of 1, 1/10, and 1/100 in each lane indicate relative amounts of samples subjected to electrophoresis and the lanes having the same number include the same amount of albumen liquid. Interestingly, only a small amount of interferon was identified in the thin albumen.
These results suggest the possibility that a relatively large amount of recombinant proteins expressed by gene knock-in are accumulated in the thick albumen.
(3) Large Amount of Interferon Proteins Detected in Thick Albumen (Estimated to be about 5 mg/ml)
Next, the thin albumen and thick albumen were collected from a wild-type egg (NC: negative control) and eggs (KI egg 1 and 2) derived from 2 human interferon β knock-in chickens. After each sample was diluted twice, an equal amount of sample was subjected to electrophoresis using a 5-20% acrylamide gel to visualize proteins contained in the albumen by Coomassie Brilliant Blue staining (CBB Stain One, Nacalai). A result is shown in FIG. 11. Similar to the previous western blotting result, clear bands are detected at a position of about 30 kDa in 2 knock-in eggs but not in the wild-type egg, indicating that these bands are human interferon β. Further, similar to the western blotting result, these human interferon β bands are hardly detected in the thin albumen subjected to electrophoresis. A comparison of CBB stained band signals revealed that amounts of human interferon β were significantly different between the thin albumen and the thick albumen although amounts of other albumen components such as ovotransferrin and ovalbumin were almost the same, demonstrating that human interferon β expressed by knock-in at the ovalbumin gene locus was dominantly accumulated in the thick albumen.
Further, a concentration of human interferon in the albumen can be estimated by analyzing the CBB staining image. An intensity of blue color caused by CBB staining is substantially proportional to an amount of proteins. A signal concentration of human interferon β having a relative amount of 1 is compared to that of ovalbumin having a relative amount of 1/10 through a quantification analysis of NIH image to obtain a ratio between them of 1.01:1. A concentration of ovalbumin that accounts for nearly half of albumen proteins is about 50 mg/ml, thus a concentration of human interferon β is estimated to be about 5 mg/ml.
The present method achieves the expression of the exogenous gene at a very high concentration of 5 mg/ml. Further, the exogenous gene is inserted in an identical location, thus variation in an expression level is small between individuals and in the same individual. Further, the present method uses a technique to perform knock-in at a translation starting site of a gene that is actually expressing in a chicken individual, thus gene expression is not reduced by an effect of gene silencing or the like in a G2 generation or later. FIG. 12 compares amounts of interferon in the albumen of eggs that have been collected 3 times (day1, day4 and day7) over a week. The amounts of interferon in 3 eggs are compared through a quantification analysis of NIH image to obtain a ratio between them of 1:0.92:0.96, thus there is almost no variation in the concentration of interferon in the thick albumen. Further, these knock-in eggs were kept at 18° C. after collection and cracked open at the same time. This shows that the exogenous protein interferon can stably exist in the albumen over a week without undergoing significant degradation or the like.
(4) Expression of Interferon in Eggs Derived from Different Individuals (Consistency of Interferon Expression)
Eggs were obtained from 4 interferon knock-in females 3 months after they laid the first eggs and then cracked open (FIG. 23). Every egg had cloudy thick albumen. Further, eggs were obtained from 5 chickens (#584, #766, #714, #645, and #640) and the thick albumen of each egg was subjected to electrophoresis and CBB staining in the same manner as described in above (3). Interferon bands were detected in all eggs (FIG. 17). The concentrations of interferon of #584, #766, #714, #645, and #640 are compared through a quantification analysis of NIH image to obtain a ratio between them of 1.0:1.0:0.94:0.91:0.89. In this ratio, a difference between the maximum concentration and minimum concentration is within 11%, demonstrating a very stable expression as compared to a variation in secretion concentrations observed between individuals (5 μg/ml to 100 μg/ml) in Non-Patent Literature 1. Having little individual difference is advantageous for obtaining a large amount of recombinant proteins using a plurality of recombinant chickens.

Example 2

(1) Attempt of Efficiently Extracting Interferon from Thick Albumen (Applicable Solubilization Treatment)
It is found that interferon β is in the thick albumen at a high concentration. This interferon β is preferably extracted and purified from the albumen for a general use. A purification technique includes various column treatments on the basis of molecular weight and chemical properties, however proteins are preferably solubilized in an aqueous solution before being subjected to the column treatment. An aqueous solution and an insoluble matter can be separated by a centrifugal operation. Thus, studies were conducted to examine whether interferon β in the thick albumen was collected in an aqueous solution or included in an insoluble matter.
The thick albumen in an amount of 200 μl was collected and subjected to centrifugation at 20,000×g for 15 minutes to separate a white precipitate fraction from a liquid fraction (a tube 1 in FIG. 13). Electrophoresis using an acrylamide gel shows that the liquid fraction contains human interferon β (lane 1 in FIG. 14), however an amount of human interferon β is clearly less than that included in an equivalent amount of the thick albumen before separation (lane 0 in FIG. 14). Thus, it was speculated that a majority of interferon β was included in the white precipitate caused by centrifugation. In order to solve this, several attempts have been made to reduce the white precipitate and increase a yield of interferon. In FIG. 13, a thick albumen liquid in an amount of 200 μl was added in each tube and subjected to the following treatments. The thick albumen liquid is added and mixed by inversion with a 4 times volume (800 μl) of a 3M saturated arginine solution (tube 2), added with a 4 times volume (800 μl) of the 3M saturated arginine solution and subjected to ultrasonic crushing (tube 3), added and mixed by inversion with a small amount of arginine (20 mg) and filled up with PBS to 1 ml (tube 4), added and mixed by inversion with a small amount of arginine hydrochloride (20 mg) and filled up with PBS to 1 ml (tube 5), filled up with PBS to 1 ml and subjected to the ultrasonic crushing (tube 6), added and mixed by inversion with arginine hydrochloride in a saturating amount or more (200 mg) (tube 7), added with a twice volume (400 μl) of the 3M saturated arginine solution and subjected to the ultrasonic crushing (tube 8), added with a small amount of arginine hydrochloride (20 mg) and subjected to the ultrasonic crushing (tube 9), or added with a small amount of sodium chloride (40 mg) and subjected to the ultrasonic crushing (tube 10). Although the white precipitates were still observed after centrifugation at 20,000×g for 15 minutes, the amounts of the white precipitates were reduced by all of these treatments as compared to that without a treatment (tube 1). In particular, the amounts of the white precipitates were markedly reduced in the tubes 3, 6, 8, and 9, which were subjected to the ultrasonic crushing.
After supernatants were recovered, samples were prepared by adjusting their loading amounts to be equal on the basis of the original amounts of the thick albumen and subjected to electrophoresis using an acrylamide gel (FIG. 14). The lane number in FIG. 14 corresponds to the tube number in FIG. 13 except lane 0, in which the thick albumen in an equivalent amount was applied to electrophoresis without separation. Although there were some differences between lanes, lane 2 to lane 10 contained more interferon β than lane 1 of the non-treatment sample. In particular, the sample prepared by adding a 4 times volume of the 3M saturated arginine solution and performing the ultrasonic crushing (tube 3) contained a significant amount of interferon β. These results show that an amount of interferon β extracted in an aqueous solution from the thick albumen increases by a physical treatment such as the ultrasonic crushing and a chemical treatment such as adding arginine or an arginine buffer (more restrictively, a solubilization treatment of insoluble protein). The sample in tube 3 prepared by adding a 4 times volume of the 3M saturated arginine solution and performing the ultrasonic crushing was subjected to centrifugation at 20 k×g for 15 minutes to recover a supernatant. The supernatant was subjected to dialysis in PBS for 24 hours (hereinafter referred to as a thick albumen rough purification product). The thick albumen rough purification product is transparent without any precipitates, suggesting that some of the white precipitates were solubilized and transferred to the supernatant by a series of treatments.

(2) Activity of Interferon Produced in Chicken Egg.

Activity of interferon in the thin albumen, thick albumen, and thick albumen rough purification product was examined by a bioassay. HEK-blue IFN-α/β (Invivogen) is a cultured cell that secretes alkaline phosphatase by human interferon β added in a medium. Activity of human interferon β can be detected by adding a medium after reaction to an alkaline phosphatase substrate solution (Quanti-Blue; Invivogen) and examining a change in the substrate solution (a color change from red to blue for Quanti-Blue). The thin albumen, the thick albumen (the supernatant after centrifugation at 20 k×g for 15 minutes), and the thick albumen rough purification product (derived from tube 3) derived from the human interferon knock-in eggs were added to the culture media of HEK-blue IFN-α/β. Further, the thin albumen derived form a wild-type chicken egg and PBS were added to the media as a negative control and recombinant human interferon was added to the media as a positive control. The cells were cultured for 20 hours and supernatants of the culture media were added to the Quanti-Blue substrate solution to perform reactions at 37° C. for 1 hour. A result is shown in FIG. 15.
The human interferon β activity is detected in any of the thin albumen, the thick albumen centrifugation supernatant, and the thick albumen rough purification product derived from the interferon knock-in (IFN-KI) eggs, demonstrating that an unpurified knock-in egg product exhibits the interferon activity and such an activity remains after the solubilization treatment by the ultrasonic crushing or the arginine buffer. Thus, interferon derived from the chicken egg can be used as it is in the egg without a processing, after a simple processing such as a centrifugation fractionation, or after a processing such as solubilization and purification.

(3) Activity Quantification of Interferon Produced in Chicken Egg

Activity of interferon in the thick albumen was measured by a bioassay. As described in (2), the thick albumen (a supernatant obtained by the ultrasonic crushing followed by centrifugation at 20,000×g for 15 minutes) derived from a human interferon knock-in egg (collected 3 months after the first egg) was serially diluted 5 folds and 10 μl of each dilution was added to the HEK-blue IFN-α/β culture medium. As a comparison, commercially available recombinant human interferon β (Wako Pure Chemical Industries, Ltd.) in a concentration of 10 μ/ml was serially diluted 5 folds in the same manner and 10 μl of each dilution was added to the culture medium. The cells were cultured for 20 hours and supernatants of the culture media were added to the Quanti-Blue substrate solution to perform reactions at 37° C. for 1 hour. A result is shown in FIG. 18. In an upper series of reactions using supernatants of the culture media to which commercially available human interferon was added, a fourth well from the left has a color in which red and blue is mixed, whereas, in a lower series of reactions using supernatants of the media to which the thick albumen was added, an eighth well from the left has a color in which red and blue is mixed (in both dilution series, concentration decreases from left to right). Thus, the interferon activity in the thick albumen is 625 or more times higher than that of the 10 μ/ml commercially available interferon and a concentration of interferon in the thick albumen is estimated to be 6.25 mg/ml or more. About 16 ml of the thick albumen was recovered at this point, meaning that human interferon having activity equivalent to about 100 mg of the commercially available interferon could be obtained from one egg. As for price, 20 μg of the recombinant human interferon β available from Wako Pure Chemical Industries, Ltd. costs 39,000 Japanese Yen, thus 100 mg of interferon is worthy of 195,000,000 Japanese Yen (about 200 million Japanese Yen). Using the present method allows the production of a very large amount of human recombinant proteins in terms of activity.

(4) Analysis of Egg of Knock-In Chicken in G2 Generation

A G1 knock-in chicken (male) was mated with a wild-type female to establish G2 knock-in chickens (male and female). The G2 knock-in chickens were raised to sexual maturity to obtain eggs and the eggs were cracked open (right in FIG. 19). All obtained eggs had cloudy thick albumen as was the case for the egg derived from G1. Further, eggs were obtained from 3 G2 knock-in chickens and their thick albumen was subjected to electrophoresis. The thick albumen of the G1 derived egg was also subjected to electrophoresis for comparison. In a CBB staining image, interferon signals were detected in the G2 derived eggs as in the G1 derived egg. These results showed that the exogenous gene knocked-in at the oviduct gene could be stably expressed in the chicken egg over generations. This observation can guarantee a large-scale and long-term stable operation of the production of recombinant proteins using a knock-in chicken.

Example 3

An egg was obtained from the chicken in which the human antibody gene was knocked-in at the human ovalbumin gene locus in a manner as described in production example 3. The presence of a human antibody protein in the albumen was examined. The albumen was added with an equal volume of a sample buffer (0.125M Tris pH6.8, 4% SDS, 10% glycerol, 0.1% BPB, note that 2-ME is not included). These samples were serially diluted 10 folds 3 times, separated by electrophoresis using a 5-20% acrylamide gel, and transferred to a PVDF membrane. After the membrane was blocked from a non-specific binding of an antibody molecule by skim milk, the membrane was subjected to western blotting using an anti-human immunoglobulin antibody (Jackson Immuno Research, Anti-Human IgG F(ab)) diluted 1,000 times as a primary antibody and an anti-rabbit HRP conjugated antibody (Jackson Immuno Research, Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L)) diluted 1,000 times as a secondary antibody. A result is shown in FIG. 20.
The antibodies detected bands of about 200 kDa at the same position as that of a purified recombinant human antibody (trade name: Herceptin, Roche Ltd.) (right in FIG. 20). Further, this band was not detected in a wild-type egg at all (left in FIG. 20). These results suggest that a human antibody complex maintains a normal subunit structure in the chicken egg. In FIG. 20, numbers of 1/20, 1/200, and 1/2 k (=1/2000) in each lane indicate relative amounts of the samples subjected to electrophoresis when the undiluted albumen is taken as 1. A concentration of the antibody complex is estimated to be 1 mg/ml or more by comparison with a loaded amount of Herceptin of a known concentration (right in FIG. 20).

Example 4

(1) Ovomucoid Homozygous Gene Knock-Out Chicken

Ovomucoid is an albumen protein, however an expression dynamic of ovomucoid in an early developmental process or a function of ovomucoid in development has not been studied. An effect of loss of function of ovomucoid is completely unknown. As shown in production example 1-3, the inventors created the heterozygous ovomucoid knock-out chickens (male and female) using the genome editing technique. Further, the inventors established the chicken having a complete deletion of ovomucoid (the homozygous ovomucoid knock-out chicken) by mating the heterozygous chickens. As shown in FIG. 16, the male and female heterozygous knock-out chickens having the same 5-base deletion in an exon 3 encoding the ovomucoid protein were mated with each other after sexual maturity to obtain offsprings. As shown in FIG. 16, the offsprings included a homozygous knock-out chicken in addition to a wild-type and heterozygous knock-out chickens. Further, both male and female ovomucoid homozygous knock-out chickens were obtained and they have been growing healthily like a wild-type chicken without any morphological abnormalities. These results showed, for the first time, that loss of function of ovomucoid did not cause any effect on an initial development such as lethality or morphological abnormalities.

(2) Usefulness of Ovomucoid Knock-Out Chicken Egg

The homozygous ovomucoid knock-out chicken does not secrete ovomucoid and thus produces an egg having no ovomucoid. Ovomucoid is a very strong allergen substance and it is known that allergenicity of ovomucoid is not lost by heating or enzymatic degradation. Needless to say, an ovomucoid deficient egg does not exhibit strong allergenicity caused by ovomucoid, thus it is clearly understood that the ovomucoid deficient egg is useful, as a low allergenic egg, for significantly reducing allergenicity in all products that use an egg, such as a raw food, a processed food, a vaccine produced in an egg, and a cosmetic raw material.

(3) Characteristics of Ovomucoid Knock-Out Chicken Egg

The homozygous knock-out female can produce an egg as it produced an egg almost every day at least for 6 months in a manner similar to that of a wild type. An image of a cracked open egg is shown in a left panel of FIG. 21. The knock-out egg is not visually different from a wild-type egg. Further, the knock-out egg is not markedly different from a normal chicken egg in processability as, for example, it is coagulated by heating (right in FIG. 21).
Further, a chicken individual can be produced by mating the homozygous knock-out chickens with each other. An egg, which was obtained by mating 5 bp-deletion homozygous male and female individuals with each other, was incubated to obtain an ovomucoid 5 bp-deletion homozygous individual in the G3 generation (FIG. 23). This result shows that a chicken can develop without the ovomucoid gene or the ovomucoid protein, that is, ovomucoid is not essential for the development of chicken.

INDUSTRIAL APPLICABILITY

Further Improvement in Expression Level of Knock-In Gene
Interferon β obtained in the present example had a very high concentration of 5 mg/ml, however a concentration of a recombinant protein in an egg can be further increased.

1. Homozygousing Knock-In Gene

The analyzed chicken egg was produced from the parental chickens having a genotype in which human interferon β was inserted into one allele of the ovalbumin gene locus (heterozygous gene knock-in). It is possible to obtain an individual that expresses a recombinant protein at a higher concentration by mating heterozygous gene knock-in parents with each other or mating germ line chimeric individuals having knock-in primordial germ cells with each other to create an individual in which human interferon β is inserted into both alleles (homozygous gene knock-in).

2. Improving Signal Peptide and Codon Usage

In the present example, human interferon β cDNA was knocked-in at the translation starting site of the ovalbumin gene. In this case, a signal peptide in use was from human interferon β, and it was not optimized for a chicken oviduct cell. Examples of the signal peptide that allows secretion in the chicken oviduct at a high efficiency include a signal peptide of a protein that is actually secreted from the oviduct. Specific examples of such a signal peptide include “MRSLLILVLCFLPLAALG” of albumen lysozyme and “MKLILCTVLSLGIAAVCFA” of ovotransferrin. Further, the present invention is not limited thereto and an artificial or natural signal peptide that allows protein secretion in a chicken cell at a high efficiency may be used. Knock-in of a desired protein having these signal peptides at its N terminus at the ovalbumin gene locus allows the production of the desired protein at a higher concentration. Further, protein production can be improved by optimizing a base sequence of cDNA to be knocked-in in accordance with a codon usage used in a chicken.

3. Increasing Copy Number of Inserted Gene

In the present example, only one gene was knocked-in, however a plurality of genes can be knocked-in in a tandem form that allows transcription and translation to simultaneously express the plurality of genes, thereby increasing their expression levels. The plurality of genes may be composed of a single gene or a plurality of kinds of genes and the number of genes may be any number. Specifically, when performing gene knock-in, a plurality of genes may be inserted by interposing a sequence such as IRES between them to facilitate transcription and translation and increase gene products. Further, a plurality of proteins may be arranged by interposing a 2A peptide or the like between them and simultaneously expressed under control of the ovalbumin promoter. In this manner, a larger amount of proteins can be produced by cleaving the peptides.
As a preferable embodiment, the light chain gene and heavy chain gene of the human antibody gene and α1 and α2 of the human type I collagen gene are each tandemly connected via the 2A peptide gene and knocked-in into a chicken.

4. Usefulness of Knock-Out Chicken Egg

The homozygous ovomucoid knock-out chicken does not secrete ovomucoid and thus produce an egg having no ovomucoid. Ovomucoid is a very strong allergen substance and it is known that allergenicity of ovomucoid is not lost by heating or enzymatic degradation. Needless to say, an ovomucoid deficient egg does not have strong allergenicity caused by ovomucoid, thus it is obvious that the ovomucoid deficient egg is useful, as a low allergenic egg, for significantly reducing allergenicity in all products that use an egg, such as a raw food, a processed food, a vaccine produced in an egg, and a cosmetic raw material. Further, an exogenous gene can be expressed in an albumin allergen knock-out chicken egg by breeding or using a genome edited primordial germ cell, and it is clearly understood that purification of the exogenous gene product produced in this manner can simplify an allergen removing process.
A poultry egg in which an oviduct-specific gene other than ovomucoid is knocked-out is useful for significantly reducing allergenicity as is the case for ovomucoid.

Claims

1-22. (canceled)

23. A genetically modified chicken egg whose genome comprises a deletion, a substitution, or an insertion of a base or bases in a coding region of an endogenous ovomucoid gene, wherein the chicken egg that does not functionally express endogenous ovomucoid.

24. The chicken egg of claim 23, wherein the coding region is exon 3 of ovomucoid gene.

25. The chicken egg of claim 24, wherein the coding region is OVMtg2 represented by the nucleotide sequence of SEQ ID NO: 6.

26. A genetically modified chicken whose genome comprises a deletion, a substitution, or an insertion of a base or bases in a coding region of an endogenous ovomucoid gene, wherein the chicken is capable of laying an egg that does not functionally express endogenous ovomucoid.

27. The chicken of claim 26, wherein the coding region is exon 3 of ovomucoid gene.

28. The chicken egg of claim 27, wherein the coding region is OVMtg2 represented by the nucleotide sequence of SEQ ID NO: 6.

29. A method of producing a genetically modified knock-out chicken egg, the method comprising:

a) deleting, substituting, or inserting a base or bases in a coding region of an endogenous ovomucoid gene of an isolated chicken primordial germ cell (PGC), wherein:

the endogenous ovomucoid gene is inactivated;

b) transplanting the PGC obtained in step a) into a recipient chicken embryo;

c) producing a genetically modified knock-out male chicken and a genetically modified knock-out female chicken from the chicken embryo in step b);

d) mating the genetically modified knock-out male chicken and the genetically modified knock-out female chicken;

e) obtaining an offspring knock-out chicken, wherein the offspring knock-out chicken is capable of laying an egg; and

f) obtaining a genetically modified knock-out chicken egg from the knock-out offspring chicken of step e), wherein the chicken egg does not functionally express endogenous ovomucoid.

30. The method of claim 29, wherein the coding region is exon 3 of ovomucoid gene.

31. The method of claim 30, wherein the coding region is OVMtg2 represented by the nucleotide sequence of SEQ ID NO: 6.

32. The method of claim 29, wherein the base or bases in the coding region of the endogenous ovomucoid gene in step a) is deleted, substituted, or inserted using CRISPR and guide RNA.