Embryo acquisition method

Abstract

Provided is a method for acquiring mammalian embryos having a high fecundation rate.　The embryos are acquired via step a) and step b), as follows.　a) A step in which a trifunctional purine biosynthetic protein adenosine-3 homolog protein (GART) and / or a gene encoding GART is introduced into a fertilized mammalian ovum. b) A step in which the fertilized ovum is cultured.

Description

How to obtain embryos

[0001] The present invention relates to a method for obtaining embryos by culturing fertilized mammalian eggs.

[0002] Reproduction by artificial insemination and in vitro fertilization is performed in various mammals, including humans and livestock. However, for example, the conception rate of artificial insemination in cattle has been declining year by year, and attempts have been made to improve the quality of fertilized eggs and embryos in order to increase this conception rate. Patent Document 1 discloses a technique for increasing the embryo development rate by in vitro culturing fertilized eggs produced by in vitro fertilization using a specific medium. Non-Patent Document 1 reports that irradiating near-infrared light on fertilized cattle eggs produced by in vitro fertilization promoted their development.

[0003] International Publication No. 2015 / 056727

[0004] Yokoo Masaki et al., Abstracts of the Japanese Society of Reproductive Biology 108(0), p.55 (2015)

[0005] An object of the present invention is to provide a method for obtaining mammalian embryos with a high conception rate.

[0006] The present invention provides the following: (1) A method for obtaining an embryo, comprising the following steps a) and b): a) introducing trifunctional adenosine-3 homolog protein (GART) and / or a gene encoding GART into a mammalian fertilized egg; and b) culturing the fertilized egg. (2) The method for obtaining an embryo according to (1), wherein the GART is a polypeptide consisting of an amino acid sequence set forth in any of SEQ ID NOS: 1 to 6, or an amino acid sequence having 90% or more sequence identity to the amino acid sequence and having GART activity. (3) The method according to (1) or (2), wherein the culture time in step b) is 16 to 216 hours. (4) A method for producing a mammalian individual, comprising the step of implanting an embryo obtained by the method according to any of (1) to (3) into a female individual and fertilizing the embryo. (5) An expression cassette for introducing a gene into a mammalian fertilized egg, comprising a gene encoding GART. (6) An expression vector for gene transfer into a mammalian fertilized egg, comprising the expression cassette according to (5). This specification incorporates the disclosure of Japanese Patent Application No. 2024-143433, from which the present application claims priority.

[0007] According to the present invention, it is possible to obtain mammalian embryos with a high conception rate.

[0008]

[0033] Figure 1 is a schematic diagram showing the purine nucleotide synthesis pathway. Figure 2 is a box plot showing the number of offspring per individual C57BL / 6 mouse and DBA / 2 mouse in Test Example 1. In the figure, "**" indicates that p<0.01 was found in a t-test (n=6). Figure 3 is a schematic diagram showing the developmental stages of mice from fertilization to the blastocyst stage and the time from fertilization to each developmental stage. Figure 4 is a bar graph showing the development rates of C57BL / 6 mice and DBA / 2 mice up to the 4-cell stage, morula stage, and blastocyst stage in Test Example 1. In the figure, error bars indicate standard error, and "**" indicates that p<0.01 was found in a t-test (n=7). Figure 4 is a box plot showing the number of ovulations per individual C57BL / 6 mouse and DBA / 2 mouse in Test Example 1. In the figure, "**" indicates that p<0.01 was found in a t-test (n=10). These are graphs and a schematic diagram showing the results of comprehensive metabolome analysis of C57BL / 6 mouse fertilized eggs and DBA / 2 mouse fertilized eggs in Test Example 2, and a comparison of the amounts of glycine metabolism-related substances between the two mice. In the bar graphs in the figures, black bars indicate the amounts of each metabolic-related substance in C57BL / 6 mice, and white bars indicate the amounts of each metabolic-related substance in DBA / 2 mice. These are graphs showing the results of a comparison of the expression levels of annotation genes in Test Example 3, comparing RNA-seq data of mouse fertilized eggs immediately after fertilization with RNA-seq data of fertilized eggs at the 2-cell stage. (A) shows the changes in genes related to the carbohydrate metabolic pathway, (B) the TCA cycle, (C) the lipid metabolic pathway, and (D) the glycine metabolic pathway. The values ​​on the vertical and horizontal axes of each graph are Logarithmic. １０The figures show the expression levels of the Gart gene in fertilized eggs of C57BL / 6 mice and DBA / 2 mice in Experimental Example 4. The expression levels of the Gart gene are shown as relative expression levels to the GAPDH gene, an endogenous control gene. In the figure, error bars indicate standard error, and "*" indicates p<0.05 in the t-test (n=4).

[0033] Figure 1 shows time-lapse micrographs of C57BL / 6 mouse fertilized eggs cultured in a medium containing lometrexol hydrate, a GART inhibitor (Gart KD), and in a medium without lometrexol hydrate (control) in Test Example 5. Figure 2 shows line graphs showing the development rates of C57BL / 6 mouse fertilized eggs to the 2-cell stage, 4-cell stage, morula stage, and blastocyst stage in Test Example 5, cultured in a medium containing lometrexol hydrate, a GART inhibitor (Gart KD), and in a medium without lometrexol hydrate (control). In the figure, error bars indicate standard error, and "*" and "****" indicate p<0.05 and p<0.0001, respectively, in a t-test (n=8). Figure 3 shows a schematic diagram of the exon / intron structure of the Gart gene and the guide RNA design positions in Test Example 6. 1 is a line graph showing the development rates of GART knockout fertilized eggs (Gart KO) and control fertilized eggs up to the 2-cell stage, 4-cell stage, morula stage, and blastocyst stage in Test Example 6. In the figure, error bars indicate standard error, and "****" indicates p<0.0001 in a t-test (n=4). This is a schematic diagram showing the procedure for the transplantation test of Gart KO and control fertilized eggs into mice in Test Example 6. Embryos cultured to the 2-cell stage from Gart KO and control fertilized eggs were transplanted via the oviduct into C57BL / 6 pseudopregnant female mice (foster mother mice), and viability to offspring was investigated. This is a boxplot showing the birth rates of mice transplanted with Gart KO and control fertilized eggs in Test Example 6.In the figure, "****" indicates that p<0.0001 in the t-test (n=14). This is a schematic diagram showing the structure of the linear DNA fragment introduced into fertilized eggs in Test Example 7. The linear DNA fragment contains, in order from the 5' end, a CAG promoter, a human GART gene, and an SV40 polyA sequence. In Test Example 7, this is a box-and-whisker plot showing the incidence of various fertilized eggs when fertilized eggs introduced with various concentrations of the human GART gene were cultured with controls. In the figure, "****" indicates that p<0.0001 in the t-test (n=5). In Test Example 8, this is a box-and-whisker diagram showing the expression levels of GART in human oocytes, fertilized eggs, 2-cell embryos, blastocysts, and ES cells analyzed based on the single-cell RNA-seq database. "PRKM" on the vertical axis stands for "Reads per kilobase of exon per million."

[0009] [1] Overview and Definition: Fertilized eggs obtained by artificial insemination of mammals, even if eggs and sperm from the same parents are used, will develop into blastocysts for implantation into the mother, while others will stop developing midway through early development. In other words, there are high-quality fertilized eggs that can lead to subsequent pregnancy and birth, and low-quality fertilized eggs that cannot. In particular, it tends to be difficult to obtain high-quality fertilized eggs as the parents age.

[0010] In order to explore factors for obtaining high-quality fertilized eggs / embryos, the present inventors performed comprehensive metabolomic analysis on fertilized eggs from C57BL / 6, a mouse species known for its high fertility rate, and DBA / 2, a mouse species known for its low fertility rate. As a result, they found that glycine and its related metabolic intermediates are significantly more abundant in the mouse species with a high fertility rate. They also found that the expression of glycinamide ribonucleotide transformylase (GART) (also known as "trifunctional adenosine-3 homolog protein"), which is involved in glycine metabolism, is enhanced during embryonic development, and that GART is involved in embryonic development and implantation. Based on these findings, the present inventors discovered that introducing a GART gene into fertilized eggs and culturing them increases the rate of embryonic development from the fertilized eggs, thereby completing the present invention.

[0011] As used herein, "mammal" refers to warm-blooded vertebrates, including primates such as humans and monkeys, rodents such as mice, rats, and rabbits, pets such as dogs and cats, and livestock such as cows, horses, pigs, and sheep. The methods of the present invention are typically used on non-human mammals (mammals other than humans). In the present invention, "human" refers to Homo sapiens. "Monkey" refers to non-human animals classified in the order Primates. "Mouse" refers to Mus musculus. "Rat" refers to Rattus norvegicus. "Rabbit" refers to animals classified in the family Leporidae. "Dog" refers to animals classified in the genus Canis lupus, typically Canis lupus familiaris. "Cat" refers to animals classified in the genus Felis silvestris, typically Felis silvestris catus. "Cow" refers to animals classified in the genus Bos, typically Bos taurus and Bos indicus. "Horse" refers to Equus caballus. "Pig" refers to animals classified as Sus scrofa, typically Sus scrofa domesticus. "Sheep" refers to Ovis aries.

[0012] As used herein, "trifunctional adenosine-3 homolog protein (GART)" refers to an enzyme encoded by the GART gene that catalyzes the three reactions represented by the following formulas (I) to (III). However, the individual enzymatic activity domains within GART that catalyze the reactions represented by the following formulas (I), (II), and (III) may also be referred to as Gas synthase, GAR formyltransferase, and AIR synthase, respectively.

[0013] GART is an enzyme that plays an important role in three reactions in the purine nucleotide synthesis pathway shown in Figure 1. The purine nucleotide synthesis pathway shown in Figure 1 is a pathway in which inosine monophosphate (IMP), the basis for adenine monophosphate (AMP) and guanine monophosphate (GMP), is synthesized from D-ribose-5-phosphate (R5P) supplied from the pentose phosphate pathway (HMS). The 1'-OH group of R5P is pyrophosphorylated to form 5-phosphoribosyl-1α-diphosphate (PRPP). Purine nucleotides are produced by assembling purine skeletons one after another using PRPP as a base. This is called de novo synthesis of nucleotides. The purine skeleton is made up of glutamic acid (Gln), glycine (Gly), aspartic acid (Asp), N10-formyl-tetrahydrofolate (THF), and CO. ２ The purine nucleotide synthesis pathway includes D-ribose-5-phosphate (R5P), 5-phosphoribosyl-1α-diphosphate (PRPP), 5-phospho-β-ribosylamine (5PRA), glycinamide ribotide (GAR), trifunctional adenosine-3 homolog protein (GART), 10-formyltetrahydrofolate (10-formylTHF), tetrahydrofolate (THF), formylglycinamide ribotide (FGAR), and formylglycine These include 5-aminoimidazole ribotide (FGAM), 5-aminoimidazole ribotide (AIR), 4-carboxy-5-aminoimidazole ribotide (CAIR), 5-aminoimidazole-4-(N-succinocarboxamide) ribotide (SACAIR), 5-aminoimidazole-4-carboxamide ribotide (AICAR), 5-formylaminoimidazole-4-carboxamide ribotide (FAICAR), and inosine monophosphate (IMP).

[0014] As used herein, fertilized eggs are cultured in a fertilized egg culture medium, preferably continuing until they develop to the blastocyst stage. The term "fertilized egg culture medium" refers to a medium commonly used for culturing fertilized eggs, such as KSOM medium, KSOMaa medium, mWM medium, BMOC2 medium, M16 medium, M12 medium, MTF medium, CZB medium, KSMO medium, mSOF medium, mSOFaa medium, G1 medium, G2 medium, TYH medium, HTF medium, mHTF medium, CR1aa medium, mCR1aa medium, TCM199 medium, and PZM5 medium.

[0015] [2] Method for Obtaining Embryos The first embodiment of the present invention is a method for obtaining embryos. The method of this embodiment is characterized by comprising the following steps a) and b): a) introducing trifunctional adenosine-3 homolog protein (GART) and / or a gene encoding GART into a mammalian fertilized egg; and b) culturing the fertilized egg.

[0016] [2-1] Step a) GART Gene Introducing Step The method of this embodiment includes the step of introducing GART and / or a gene encoding GART into a fertilized egg. In the present specification, GART can be a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, or an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence, and having GART activity.

[0017] The polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is GART derived from humans. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 is GART derived from bovines. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 is GART derived from equines. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 is GART derived from porcines. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 is GART derived from ovines. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 is GART derived from mice. The amino acid sequences of SEQ ID NOs: 1 to 6 are shown in Table 1.

[0018]

[0019] In this step, it is particularly preferable to introduce the GART gene into a fertilized egg. In this case, the GART gene may be introduced into the fertilized egg as an expression cassette. As used herein, the expression cassette is not particularly limited as long as it is DNA having a structure capable of expressing the GART gene in cells. The expression cassette may have a structure including, for example, a promoter and a GART gene placed under the control of the promoter. The promoter is not particularly limited, and for example, various Pol II promoters can be used. Pol II promoters are not particularly limited, and examples thereof include a CAG promoter, a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, and a β-actin promoter.

[0020] The expression cassette may contain a signal sequence in addition to the above. It may also contain other sequences useful for promoting the expression or activity of GART. A signal sequence refers to a base sequence encoding all or part of a protein that can increase the translation efficiency of a target gene encoding a target protein. Any known signal sequence may be used, for example, a polyadenylation (polyA) signal operably linked to the nucleic acid coding sequence from the 3' side, such as an SV40 polyA signal or a Chinook salmon polyA signal.

[0021] The GART gene or an expression cassette containing the GART gene is preferably transiently introduced into the fertilized egg without being integrated into the genome. The form of the expression cassette and the method of introduction are not particularly limited, as long as the expression cassette can be transiently introduced into the fertilized egg. For example, the expression cassette can be introduced as a linear DNA fragment or circular DNA by microinjection, electroporation, calcium phosphate precipitation, or a biolistic method (e.g., tungsten bombardment), or directly by contacting a naked nucleic acid vector or construct with cells in a solution.

[0022] The expression cassette may be introduced into a fertilized egg via a vector. In this case, it is preferable to transiently introduce the GART gene so that the introduced gene is not incorporated into the genome of the fertilized egg. In this case, for example, a method of introducing the expression cassette into the fertilized egg using an adenovirus vector, an adeno-associated virus vector, or the like, which are known as transient gene vectors, may be used.

[0023] Instead of or in addition to the GART gene, GART itself may be introduced into the fertilized egg. The introduction of GART into the fertilized egg can be carried out by a known protein introduction method, such as a cationic lipid-type protein introduction reagent, a membrane-permeable peptide-type protein introduction reagent, a Sendai virus-derived envelope, microinjection, electroporation, etc.

[0024] [2-2] Step b) Fertilized Egg Culturing Step In step b) of the method of this embodiment, the conditions such as the medium, temperature, and atmospheric gas composition used for culturing the fertilized eggs (embryos) can be those commonly used depending on the mammalian species. For example, when used with bovine fertilized eggs, mSOF medium, mSOFaa medium, CR1aa medium, mCR1aa medium, TCM199 medium, etc. can be used. The culture temperature can be, for example, 36.0 to 40.0°C, preferably 38.5°C. Furthermore, the culture can be carried out, for example, at saturated humidity and 5% CO ２ / 95% air, saturated humidity, 5% CO ２ / 5% O ２ / 90%N ２ The culture can be carried out under the following conditions: The culture time is preferably 16 to 216 hours, and more preferably 24 to 168 hours.

[0025] In the method of this embodiment, the medium used in step b) may contain at least one of glycine, β-alanine, and a glycine metabolism-related substance. The glycine metabolism-related substance is a compound related to the purine nucleotide synthesis pathway shown in FIG. 1, specifically, inosine monophosphate (IMP), D-ribose-5-phosphate (R5P), 5-phosphoribosyl-1α-diphosphate (PRPP), 5-phospho-β-ribosylamine (5PRA), glycinamide ribotide (GAR), trifunctional adenosine-3 homolog protein (GART), 10-formyltetrahydrofolate (10-formylTHF), tetrahydrofolic acid (THF), formylglycinamide ribonucleotide (PGRI), and the like. The compound may be at least one selected from the group consisting of 5-aminoimidazole ribotide (FGAR), formylglycine amidine ribotide (FGAM), 5-aminoimidazole ribotide (AIR), 4-carboxy-5-aminoimidazole ribotide (CAIR), 5-aminoimidazole-4-(N-succinocarboxamide) ribotide (SACAIR), 5-aminoimidazole-4-carboxamide ribotide (AICAR), and 5-formylaminoimidazole-4-carboxamide ribotide (FAICAR). In particular, the compound may be at least one selected from the group consisting of compounds generated by a reaction mediated by trifunctional adenosine-3 homolog protein (GART), specifically, IMP, AIR, CAIR, SACAIR, AICAR, and FAICAR. Furthermore, the compound may be IMP.

[0026] When glycine and / or β-alanine are added to the medium, the concentration of the added glycine and / or β-alanine can be 0.0001 to 50 mM. For example, the glycine and / or β-alanine concentration in a medium for culturing mouse fertilized eggs can be 0.1 to 2 mM. For example, the glycine and / or β-alanine concentration in a medium for culturing bovine fertilized eggs can be 1 to 50 mM.

[0027] When IMP is added to the medium as a glycine metabolism-related substance, the concentration of IMP in the medium can be 0.1 to 10 nM, particularly 0.5 to 5 nM, 0.8 to 1.2 nM, or even 1 nM.

[0028] The embryo obtained by the method of this embodiment can be developed into a new individual (baby), particularly after it has developed into a blastocyst, by being implanted into a female individual and fertilized.

[0029] [3] Method for Producing Mammalian Individuals A second embodiment of the present invention is a method for producing mammalian individuals. The method of this embodiment is characterized by including a step of implanting an embryo obtained using the method described in "[2] Method for Obtaining Embryos" into a female individual to cause fertilization. According to the method of this embodiment, it is possible to increase the conception rate and birth rate, and to obtain mammals more efficiently.

[0030] In this embodiment, the mammalian individual to be produced is preferably a non-human animal, particularly a cow, pig, horse, mouse, rat, dog, cat, goat, or sheep.

[0031] In the method of this embodiment, the female individual (recipient) receiving the transplant is in a pseudopregnant state (luteal phase) or in estrus, and the embryo is transplanted into the oviduct or the like of the female individual. After conception, the steps of obtaining offspring and growing the offspring to obtain individuals can be carried out by conventional methods.

[0032] [4] GART Gene Expression Cassette A third embodiment of the present invention is an expression cassette for introducing a gene into a mammalian fertilized egg. The expression cassette of this embodiment is characterized by comprising a gene encoding GART.

[0033] In this embodiment, GART can be a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence, and having GART activity.

[0034] In this embodiment, the expression cassette is not particularly limited as long as it is DNA having a structure capable of expressing the GART gene in cells. The expression cassette may have a structure including, for example, a promoter and the GART gene placed under the control of the promoter. The promoter is not particularly limited, and various Pol II promoters can be used. Examples of Pol II promoters include, but are not limited to, the CAG promoter, the CMV promoter, the EF1 promoter, the SV40 promoter, the MSCV promoter, the hTERT promoter, and the β-actin promoter.

[0035] The expression cassette may contain a signal sequence in addition to the above. It may also contain other sequences useful for promoting the expression or activity of GART. A signal sequence refers to a base sequence encoding all or part of a protein that can increase the translation efficiency of a target gene encoding a target protein. Any known signal sequence may be used, including, for example, a polyadenylation (polyA) signal operably linked to the nucleic acid coding sequence from the 3' end, such as an SV40 polyA signal or a Chinook salmon polyA signal. The expression cassette may also contain elements such as a polycistronic sequence for simultaneous expression with other genes, such as an IRES sequence or a 2A sequence, and an expression reporter gene such as GFP or tdTomato.

[0036] The gene expression cassette of this embodiment can improve the development rate and conception rate of fertilized eggs by introducing it into fertilized eggs, particularly low-quality fertilized eggs with low development rate and conception rate.

[0037] [5] GART Gene Expression Vector A fourth embodiment of the present invention is a GART gene expression vector. The vector of this embodiment is characterized by including the expression cassette described in "[4] GART Gene Expression Cassette."

[0038] The vector used in this embodiment is preferably a vector that transiently transfers the GART gene so that the introduced GART gene is not incorporated into the genome of the fertilized egg. In this case, for example, an adenovirus vector, an adeno-associated virus vector, or the like, which are known as transient gene vectors, may be used.

[0039] By using the gene expression vector of this embodiment, a gene expression cassette can be introduced into fertilized eggs, particularly low-quality fertilized eggs with low development and conception rates, thereby making it possible to improve the development and conception rates of the fertilized eggs.

[0040] EXAMPLES Hereinafter, examples will be shown to explain the present invention in more detail, but it is not intended that the scope of the present invention be limited to the scope of the examples.

[0041] [Test Example 1] Relationship between Mouse Species and Litter Size, Oocyte Size, and Developmental Number (1) Comparison of Litter Size C57BL / 6 mice, which are considered to have good embryo quality, and DBA / 2 mice, which are considered to have poor embryo quality, were naturally mated, and the number of offspring per individual was counted (n=6). The results of comparing the litter size of each mouse species are shown in Figure 2. In the figure, error bars indicate standard error, and "**" indicates that p<0.001 in the t-test. As shown in the figure, it was confirmed that C57BL / 6 mice produced significantly more offspring.

[0042] (2) Comparison of Egg Number and Development Rate Eight-week-old female C57BL / 6 mice and DBA / 2 mice were injected with PMSG and hCG to induce superovulation. Eggs were collected from each mouse group and fertilized in vitro. The resulting fertilized eggs were cultured in KSOM medium for up to 96 hours, and the number of eggs that developed into blastocysts was counted ( FIG. 3 ). This experiment was repeated seven times. The number of ovulated eggs was also counted. Each mouse group consisted of 10 mice. The number of ovulations for each mouse type is shown in FIG. 5 . The rate at which fertilized eggs developed into blastocysts for each mouse type is shown in FIG. 4 . In the figures, error bars indicate standard error, and "**" indicates that p<0.001 in the t-test. It was confirmed that the number of ovulations was significantly higher in DBA / 2 mice. On the other hand, the rate at which fertilized eggs developed to the blastocyst stage was approximately 90% in C57BL / 6 mice, but less than 20% in DBA / 2 mice.

[0043] Test Example 2: Comprehensive Metabolomic Analysis 3,259 C57BL / 6 mouse fertilized eggs and 1,587 DBA / 2 mouse fertilized eggs cultured in KSOM medium for 24 hours after fertilization were each dissolved in methanol. Comprehensive metabolomic analysis was performed on each solution using a CE-TOFMS (Agilent CE-TOFMS system) to measure the metabolites contained. The results of a comparison of the components of C57BL / 6 mouse fertilized eggs and DBA / 2 mouse fertilized eggs are shown in Figure 6. In the figure, black bars represent the amount of each component in C57BL / 6 mouse fertilized eggs, and white bars represent the amount of each component in DBA / 2 mouse fertilized eggs. Comparison of metabolites between the two mouse species revealed that the content of glycine and its related metabolic intermediates was significantly lower in DBA / 2 mouse fertilized eggs.

[0044] [Test Example 3] Analysis of Gene Expression in Fertilized Eggs RNA-seq data of mouse fertilized eggs immediately after fertilization in the public database (National Center for Biotechnology Information (NCBI)) was compared with RNA-seq data of fertilized eggs at the 2-cell stage, and the expression levels of annotation genes were compared. Figure 7 shows the results of comparing the expression levels of annotation genes related to each metabolic system in fertilized eggs and 2-cell stage embryos. Figure 7A shows the fluctuations in genes related to the carbohydrate metabolic pathway, Figure 7B shows the TCA cycle, Figure 7C shows the lipid metabolic pathway, and Figure 7D shows the fluctuations in genes related to the glycine metabolic pathway. The values ​​on the vertical and horizontal axes of each graph are Logarithmic. １０ The figures show the transcripts per million (TPM). The TPM (transcripts per million) value is the number of fragments per million mapped fragments, assuming all genes are the same length, and indicates the expression level of a gene in a target cell. Dots above the diagonal line of each graph indicate genes whose expression increases with development. Dots below the diagonal line indicate genes whose expression level decreases with development. In other metabolic systems, both genes whose expression level increases and genes whose expression level decreases are observed, but in the glycine metabolic system, it was confirmed that the expression of all related genes increases.

[0045] [Test Example 4] Analysis of Embryonic Development-Related Genes To investigate which glycine metabolism-related genes are particularly associated with embryonic development, total RNA was extracted from 100 C57BL / 6 mouse fertilized eggs and 100 DBA / 2 mouse fertilized eggs, and real-time PCR was performed to measure the gene expression levels. As a result, it was found that the GART gene expression level was significantly higher in C57BL / 6 mouse fertilized eggs compared to DBA / 2 mouse fertilized eggs. Figure 8 shows the expression level of the GART gene in each mouse species. The GART gene expression level is shown as the relative expression level to the GAPDH gene, an endogenous control gene. In the figure, error bars indicate standard error, and "*" indicates p<0.05 in the t-test.

[0046] Test Example 5: GART Knockdown Test To examine the effect of GART protein on embryonic development, lometrexol hydrate, a GART inhibitor, was added to the medium at a concentration of 1 μM, and C57BL / 6 mouse fertilized eggs were cultured. As a control, culture was similarly performed in medium without lometrexol hydrate. The number of fertilized eggs under each condition was 100 or more. The test was performed eight times. Figure 9 shows time-lapse micrographs of each fertilized egg from immediately after fertilization to 16 hours later. In the control (top row), the blastocyst's blastocyst cavity (hollow portion) initially shrank but expanded after 16 hours. In contrast, in the fertilized eggs treated with lometrexol hydrate, the formed blastocyst cavity disappeared, and the embryo died. Figure 10 shows the percentage of fertilized eggs that developed to each stage. In the figure, error bars indicate standard error, and "*" and "****" indicate p<0.05 and p<0.0001 in the t-test, respectively. It was confirmed that knocking down GART in fertilized eggs caused development to cease in many fertilized eggs by the blastocyst stage, with the development rate reaching nearly zero. These results indicated that GART is an essential factor for maintaining the survival and maintenance of blastocysts.

[0047] [Test Example 6] GART Knockout Test Five guide RNAs, crRNA1, crRNA2, crRNA3, crRNA4, and crRNA5, were designed based on the sequences of exons 1, 2, 3, and 19 and intron 20 of the Gart gene, which has the structure shown in Figure 11. A CRISPR-Cas9 complex (Gart KO) containing these as guide RNAs was introduced into C57BL / 6 mouse fertilized eggs, and genome editing was performed. As a control, fertilized eggs were prepared in which a CRISPR-Cas9 complex containing no guide RNA was introduced instead of the CRISPR-Cas9 complex. More than 100 fertilized eggs were prepared for each test. GART knockout fertilized eggs and control fertilized eggs were cultured for 96 hours each. The test was performed four times. During the test, the number of cells whose development had stopped was counted at each stage. Figure 12 shows the percentage of Gart KO and control fertilized eggs that developed to each stage. In the figure, error bars indicate standard errors, and "****" indicates p<0.0001 in the t-test.

[0048] Embryos from Gart KO and control fertilized eggs were cultured to the two-cell stage, and then transferred via the oviduct into pseudopregnant C57BL / 6 female mice (foster mother mice) to examine whether they would develop into offspring (Figure 13). The experiment was conducted 14 times. Two mice from each group were dissected to observe whether implantation occurred. While implantation was observed in mice receiving control embryos, implantation was not observed in the group receiving embryos derived from GART KO fertilized eggs. Figure 14 shows the birth rate for each group. In the figure, error bars indicate standard error, and "****" indicates p<0.0001 in the t-test. The birth rate for the control mice was approximately 25%, while that for the Gart KO mice was 0%.

[0049] [Test Example 7] Test for introducing GART gene into fertilized eggs. A solution (solvent: Nuclease-Free Duplex Buffer manufactured by Integrated DNA Technologies) containing an expression cassette consisting of the CAG promoter, hGART gene, and SV40 polyA sequence as shown in Figure 15 as a linear DNA fragment at concentrations of 100 ng / μL and 200 ng / μL was prepared and introduced into fertilized eggs of DBA / 2 mice by electroporation. It was previously confirmed that this linear DNA fragment was hardly integrated into the mouse genome and transiently expressed the GART protein in the fertilized eggs of DBA / 2 mice. As a control, the solvent alone was introduced into fertilized eggs of DBA / 2 mice. Table 2 shows the base sequence (SEQ ID NO: 7) of the introduced linear DNA fragment.

[0050]

[0051] Mouse fertilized eggs after introduction of the hGART gene and control fertilized eggs were cultured for up to 96 hours in KSOM medium. Figure 16 shows the development rates to the blastocyst stage of fertilized eggs introduced with the hGART gene and control fertilized eggs. In the figure, "****" indicates that p<0.0001 in the t-test (n=5). It was confirmed that the development rate to the blastocyst stage was significantly higher in fertilized eggs introduced with 100 ng / μL of DNA fragment.

[0052] [Test Example 8] Based on the single cell RNA-seq database of the public database of GART expression analysis data in human fertilized eggs (Series GSE36552, etc.), the expression levels of GART in human oocytes, fertilized eggs, 2-cell embryos, blastocysts, and ES cells were analyzed. The results are shown in Figure 17. While GART expression was barely observed in oocytes, fertilized eggs, and 2-cell embryos, GART expression was observed in preimplantation blastocysts. Furthermore, GART expression was observed in ES cells produced from blastocysts to the same extent as in blastocysts. From these results, it is speculated that the improvement in embryo development rate due to the introduction of the GART gene into fertilized eggs will have a similar effect on human embryos. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for obtaining an embryo, comprising the following steps a) and b): a) introducing trifunctional adenosine-3 homolog protein (GART) and / or a gene encoding GART into a fertilized egg of a mammal; and b) culturing the fertilized egg.

2. A method for obtaining an embryo as described in claim 1, wherein the GART is a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence and having GART activity.

3. The method according to claim 1, wherein the culture time in step b) is 16 to 216 hours.

4. A method for producing a mammalian individual, comprising the step of implanting an embryo obtained by the method according to any one of claims 1 to 3 into a female individual and causing fertilization.

5. An expression cassette for gene transfer into mammalian fertilized eggs, comprising a gene encoding GART.

6. An expression vector for gene transfer into mammalian fertilized eggs, comprising the expression cassette of claim 5.

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