Application of LincGET for improving embryonic development efficiency

Abstract

The invention relates to the field of biological medicine, and relates to a nucleic acid molecule for coding LincGET, or application of the nucleic acid molecule for coding LincGET and a nucleic acid molecule for coding Kdm4d in preparation of a medicine, and the medicine is used for improving the embryonic development efficiency of mammals. The invention also relates to a method for treating mammalian embryonic cells in vitro and an assisted reproduction method.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and relates to the use of LincGET and Kdm4d in the preparation of a medicament for improving the developmental efficiency of mammalian embryos. The present invention also relates to a method for in vitro processing of mammalian embryonic cells and an assisted reproductive method. Background Art

[0002] Intracytoplasmic sperm injection (ICSI) is a second-generation assisted reproductive technology currently used clinically. It can provide technical assistance for patients with abnormal sperm motility and low fertilization ability. Compared with in vitro fertilization (IVF), ICSI has the advantage of being able to inject sperm with low fertilization ability into embryos for fertilization.

[0003] Round spermatid injection (ROSI) is another assisted reproductive technology, but it is not widely used clinically due to its low embryo development efficiency. Compared with IVF and ICSI, ROSI can provide technical assistance for male patients with azoospermia but spherical sperm to obtain fertilized embryos, with broad application prospects and high clinical value. However, the birth efficiency of ROSI embryos is currently low, and how to improve the development and birth efficiency of ROSI embryos has become an urgent issue. Summary of the Invention

[0004] In this application, the inventors, through in-depth research, discovered that LincGET and Kdm4d can improve the developmental efficiency of mammalian reconstructed embryos, and further developed the use of LincGET and Kdm4d in the preparation of a drug for improving the developmental efficiency of mammalian reconstructed embryos. This led to the following invention.

[0005] In one aspect, the present invention provides the use of item (1) or (2) below in preparing a drug for improving the development efficiency of mammalian embryos.

[0006] (1) a nucleic acid molecule encoding LincGET; or

[0007] (2) Nucleic acid molecules encoding LincGET and nucleic acid molecules encoding Kdm4d.

[0008] In certain embodiments, item (1) or item (2) is introduced into an oocyte or embryonic cell of a mammal.

[0009] In certain embodiments, the oocyte is an oocyte in metaphase II of the meiotic division.

[0010] In certain embodiments, the embryonic cell is a two-cell stage embryonic cell.

[0011] In certain embodiments, the two-cell stage embryonic cell comprises two cells, and item (1) or item (2) is introduced into any one cell of the two-cell stage embryonic cell, or item (1) or item (2) is introduced into both cells of the two-cell stage embryonic cell.

[0012] In certain embodiments, item (1) or item (2) is introduced into an oocyte or embryonic cell of a mammal by injection.

[0013] In certain embodiments, the injection is microinjection.

[0014] In certain embodiments, the embryonic cells are obtained by round spermatid injection (ROSI), intracytoplasmic sperm injection (ICSI), or somatic cell nuclear transfer (SCNT).

[0015] In certain embodiments, the medicament further comprises one or more pharmaceutically acceptable excipients.

[0016] In certain embodiments, the one or more pharmaceutically acceptable excipients may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0017] In certain embodiments, the drug can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0018] In certain embodiments, the drug is administered in the form of a solution.

[0019] In certain embodiments, the solution comprises a nucleic acid molecule encoding LincGET, and the concentration of the nucleic acid molecule encoding LincGET in the solution is 150-350 ng / μL.

[0020] In certain embodiments, the concentration of the LincGET-encoding nucleic acid molecule in the solution is 200-300 ng / μL.

[0021] In certain embodiments, the concentration of the LincGET-encoding nucleic acid molecule in the solution is 250 ng / μL.

[0022] In certain embodiments, the medicament further comprises a nucleic acid molecule encoding Kdm4d, and the concentration of the nucleic acid molecule encoding Kdm4d in the solution is 100-200 ng / μL.

[0023] In certain embodiments, the concentration of the Kdm4d encoding nucleic acid molecule in the solution is 120-180 ng / μL.

[0024] In certain embodiments, the concentration of the Kdm4d encoding nucleic acid molecule in the solution is 150 ng / μL.

[0025] In certain embodiments, the solution is an aqueous solution.

[0026] In certain embodiments, the aqueous solution is prepared from ultrapure water.

[0027] In certain embodiments, the nucleic acid molecule encoding LincGET and / or the nucleic acid molecule encoding Kdm4d is introduced into a mammalian embryonic cell in one of the following forms:

[0028] Isolated nucleic acid molecules (eg, DNA or RNA), nucleic acid constructs, recombinant expression vectors, liposome particles.

[0029] In certain embodiments, the nucleic acid molecule encoding LincGET comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more base substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 base substitutions, deletions, or additions) thereto.

[0030] In certain embodiments, the nucleic acid molecule encoding Kdm4d comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more base substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 base substitutions, deletions, or additions) thereto.

[0031] In certain embodiments, the developmental efficiency of the embryo includes blastocyst development efficiency, embryo implantation rate or birth rate.

[0032] In the context of the present invention, the blastocyst development efficiency is determined by the ratio of the number of embryos that develop into blastocysts to the total number of embryos, as well as the expression of pluripotency marker genes (e.g., Sox2, Oct4, Nanog). The higher the proportion of embryos that develop into blastocysts, the greater the number of cells expressing pluripotency marker genes, and the higher the blastocyst development efficiency.

[0033] The embryo implantation process generally includes stages such as embryo positioning, adhesion and invasion in the endometrium, involving the growth, differentiation and migration of cells at the maternal-fetal interface, as well as common cell physiological mechanisms such as mutual recognition, adhesion, and signal transduction between cells.

[0034] In the context of the present invention, the birth rate refers to the ratio of the number of embryos that develop into mammals that are eventually born to the total number of embryos.

[0035] In certain embodiments, the mammal is one or more selected from humans, monkeys, gorillas, rats, mice, pigs, cows, sheep, and horses.

[0036] In certain embodiments, the mammal is a non-human mammal.

[0037] In another aspect, the present invention provides a method for treating mammalian embryonic cells in vitro, comprising the step of introducing the following item (1) or (2) into the mammalian embryonic cells:

[0038] (1) a nucleic acid molecule encoding LincGET; or

[0039] (2) Nucleic acid molecules encoding LincGET and nucleic acid molecules encoding Kdm4d.

[0040] In certain embodiments, the embryonic cell is a two-cell stage embryonic cell.

[0041] In certain embodiments, the two-cell stage embryonic cell comprises two cells, and item (1) or item (2) is introduced into any one cell of the two-cell stage embryonic cell, or item (1) or item (2) is introduced into both cells of the two-cell stage embryonic cell.

[0042] In certain embodiments, item (1) or item (2) is introduced into an embryonic cell of a mammal by injection.

[0043] In certain embodiments, the injection is microinjection.

[0044] In certain embodiments, the embryonic cells are obtained by round spermatid injection (ROSI), intracytoplasmic sperm injection (ICSI), or somatic cell nuclear transfer (SCNT).

[0045] In certain embodiments, item (1) or item (2) is administered in the form of a solution.

[0046] In certain embodiments, the concentration of the LincGET-encoding nucleic acid molecule in the solution is 150-350 ng / μL.

[0047] In certain embodiments, the concentration of the LincGET-encoding nucleic acid molecule in the solution is 200-300 ng / μL.

[0048] In certain embodiments, the concentration of the LincGET-encoding nucleic acid molecule in the pharmaceutical solution is 250 ng / μL.

[0049] In certain embodiments, the concentration of the Kdm4d encoding nucleic acid molecule in the solution is 100-200 ng / μL.

[0050] In certain embodiments, the concentration of the Kdm4d encoding nucleic acid molecule in the solution is 120-180 ng / μL.

[0051] In certain embodiments, the concentration of the Kdm4d encoding nucleic acid molecule in the solution is 150 ng / μL.

[0052] In certain embodiments, the solution is an aqueous solution.

[0053] In certain embodiments, the aqueous solution is prepared from ultrapure water.

[0054] In certain embodiments, the nucleic acid molecule encoding LincGET and / or the nucleic acid molecule encoding Kdm4d is introduced into a mammalian embryonic cell in one of the following forms:

[0055] Isolated nucleic acid molecules (eg, DNA or RNA), nucleic acid constructs, recombinant expression vectors, liposome particles.

[0056] In certain embodiments, the nucleic acid molecule encoding LincGET comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more base substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 base substitutions, deletions, or additions) thereto.

[0057] In certain embodiments, the nucleic acid molecule encoding Kdm4d comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more base substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 base substitutions, deletions, or additions) thereto.

[0058] In certain embodiments, the mammal is one or more selected from humans, monkeys, gorillas, rats, mice, pigs, cows, sheep, and horses.

[0059] In certain embodiments, the mammal is a non-human mammal.

[0060] In another aspect, the present invention provides a method for assisted reproduction, comprising the following steps:

[0061] According to the method for treating mammalian embryonic cells in vitro of the present invention, treated mammalian embryonic cells are obtained.

[0062] In certain embodiments, the method further comprises the step of transplanting the embryonic cells into a subject.

[0063] In certain embodiments, the embryonic cells are transplanted at the two-cell stage.

[0064] In certain embodiments, the mammal and the subject are the same individual or different individuals of the same species.

[0065] In certain embodiments, the mammal and the subject are non-human mammals.

[0066] In certain embodiments, the assisted reproductive method may be for therapeutic or non-therapeutic purposes.

[0067] Definition of terms

[0068] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0069] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0070] The terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0071] As used herein, the term "LincGET" refers to an endogenous retrovirus-associated long noncoding RNA (LTRNA). It is essential for 2-cell embryonic development and is differentially expressed in blastomeres from the late 2-cell to 8-cell embryos. LincGET forms a complex with CARM1 to promote ICM gene expression. Overexpression of LincGET establishes more H3R26me modifications within the genome, which can bias blastomeres toward the inner cell mass (ICM) fate. The sequence of LincGET is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: CH466522.1).

[0072] As used herein, the term "Kdm4d" refers to histone lysine demethylase 4D, which belongs to the JMJD2 family of histone demethylases. Its primary function is to remove methyl groups from H3K9me2 and H3K9me3. The sequence of Kdm4d is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: KU245560.1).

[0073] As used herein, the term "intracytoplasmic sperm injection (ICSI)" refers to an assisted reproductive technology that involves injecting a single sperm directly into the cytoplasm of an egg, thereby helping sperm that cannot naturally fuse and fertilize to complete the fertilization process. It is also known as the second-generation in vitro fertilization technology.

[0074] As used herein, the term "round spermatid injection (ROSI)" refers to an assisted reproductive technology in which mature sperm precursors (round spermatids) retrieved from semen or testicles are injected into the cytoplasm of an egg. Embryos obtained through ROSI can exhibit reprogramming defects during embryonic development, leading to inefficient development.

[0075] As used herein, the term "somatic cell nuclear transfer (SCNT)" refers to a technique in which a differentiated somatic cell nucleus is transferred into an enucleated oocyte to reprogram the chromatin of the somatic cell and subsequently initiate the development of the embryo into a complete individual.

[0076] The term "oocyte" as used herein refers to a gamete of a female animal. The gamete may be immature or mature.

[0077] The term "sperm" herein refers to the gametes of male animals. The sperm may be mature or immature, and at maturity, it is capable of penetrating and fertilizing an oocyte.

[0078] As used herein, the term "embryo" refers to a single-cell or multicellular organism formed after fertilization of a mammal. The embryos used in this application can be isolated from a mammal or generated by in vitro culture of mammalian stem cells under appropriate conditions, wherein the embryos generated by in vitro culture are similar to natural embryos produced in vivo at the corresponding stage, such as having similar morphology, length, weight, cell type composition, and expression of developmental marker genes.

[0079] As used herein, term "embryonic development" refers to the biological process of growth and development of mammalian embryos before they are formed into embryos with a complete organ system. Usually, embryonic development is carried out in a continuous stage, and wherein the process starts with fertilizing an egg (i.e., an ovum) with sperm (i.e., a sperm) to produce a diploid zygote. After a series of mitotic cell divisions, the zygote forms a multicellular embryo through 2-cell phases (also known as two-cell phases), 4-cell phases, 8-cell phases, and 16-cell phases. After cell division, blastocysts are formed, and then blastocysts become blastocysts. The further development of blastocysts makes it possible to form germ layers in the process called gastrulation. The subsequent stages of embryonic development include the formation of nervous system, organs, and somites.

[0080] As used herein, the term "zygote" refers to a fertilized egg or a 1-cell embryo.

[0081] As used herein, the term "blastomere" refers to a single cell in a multicellular structure formed by a fertilized egg or a one-cell embryo of a mammal after a series of mitotic divisions.

[0082] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0083] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0084] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0085] Advantageous Effects of the Invention

[0086] The present invention provides a new use of LincGET and Kdm4d for improving the developmental efficiency of mammalian embryos (e.g., ROSI embryos), thereby significantly improving the blastocyst development efficiency and birth rate of ROSI embryos, approaching the birth rate of normal embryos. The invention can be applied to the breeding of rare mammals and has important clinical value.

[0087] Sequence information

[0088] The information of the sequences involved in the present invention is described in the following table:

[0089] Table 1: Sequence description

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] DETAILED DESCRIPTION

[0099] The invention will now be described in the following non-limiting examples.

[0100] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0101] Example 1: Oocyte processing and embryo preparation

[0102] 1.1 Oocyte Collection

[0103] (1) Prepare 3 cm imported embryo culture dishes in advance, make 7 50 μL culture drops of M16 culture medium (M7292, Sigma) in each dish, and add 3-4 mL of paraffin oil to cover the culture medium.

[0104] (2) 8-week-old, healthy female mice (B6D2F1, Beijing Weitonglihua) were selected and injected with 5 IU of pregnant mare's serotonin (PMSG) (G024, Ningbo Second Hormone Factory) at 5:00 PM on the same day. The next day, 5 IU of human chorionic gonadotropin (HCG) (GN026, Ningbo Second Hormone Factory) were intraperitoneally administered. Oocytes were retrieved 15-17 hours after HCG injection, and the obtained oocytes were MII stage oocytes.

[0105] (3) The best time to perform the nuclear transfer procedure is around 9:00 AM on the second day after HCG injection. Preheat the egg retrieval solution (Hepes-CZB) and hyaluronidase (MB3094-J-100MG, Sigma) to 37°C. Place 100 μL of the preheated egg retrieval solution in a sterile culture dish and cover.

[0106] (4) Kill the mouse by dislocating the neck. Use small scissors to cut the connection between the ovary and the fallopian tube, and finally cut the fallopian tube. Place the cut fallopian tube into the egg retrieval solution prepared in step (3).

[0107] (5) Quickly place the fallopian tube under the microscope. Under the microscope, pinch the fallopian tube with sterile forceps and use the tip of fine forceps to cut open the enlarged part to allow the egg mass to flow into the liquid.

[0108] (6) Use a sterile 200 μL yellow gun tip to first aspirate a small amount of the preheated hyaluronidase in step (3), keeping the tip free of air. Then aspirate the entire egg mass into the gun tip and spit it into a 50 μL preheated hyaluronidase droplet. Let it soak for about 20 seconds. Then, use the yellow gun tip to gently blow the droplet without creating bubbles. While blowing and aspirating, observe. When all the granulosa cells on the oocyte are digested, aspirate 2 times the volume of the droplet to terminate the digestion. Gently blow and aspirate about 10 times.

[0109] (7) Gently shake the culture dish back and forth and left and right to concentrate the oocytes in the center of the droplet. Use a yellow pipette tip to transfer all the oocytes to a new 200 μL droplet of egg retrieval solution. Gently pipette about 10 times and shake gently to concentrate the eggs in the center. Repeat this step 3 times to remove the granulosa cells. The resulting oocytes are transferred to M16 medium (M7292, Sigma) for culture. The oocytes are MII stage oocytes.

[0110] 1.2 Preparation of LincGET RNA and Kdm4d RNA

[0111] 1.2.1 LincGET RNA

[0112] (1) Extraction of total RNA

[0113] Using PureLink TM RNA Mini Kit (Ambion): 300 B6D2F1 mouse late two-cell embryos were collected, 300 μL of Lysis Buffer was added, and after vortexing, an equal volume of 70% ethanol was added. 700 μL of each volume was centrifuged at 12,000 g for 15 seconds through the column. 700 μL of Wash Buffer I was then added and centrifuged at 12,000 g for 15 seconds. A new collection tube was then added, 700 μL of Wash Buffer II was added, and the tube was centrifuged at 12,000 g for 15 seconds. Wash Buffer II was then repeated. The tube was then centrifuged at 12,000 g for 15 seconds at room temperature for 2 minutes, and air-dried at 65°C for 3 minutes. The tube was then dissolved in 50 μL of water and centrifuged at 12,000 g for 1 minute. The tube was transferred to a new 1.5 ml microcentrifuge tube and the concentration was measured.

[0114] (2) Reverse transcription to obtain cDNA

[0115] Using the M-MLV Reverse Transcriptase Kit (Invitrogen), add 1 μg of RNA obtained in step (1), 1 μL of dNTP, 0.5 μL of random primer, and 0.5 μL of Oligo DT, incubate at 65°C for 5 minutes, and transfer to ice. On ice, add 1 μL of RNasin, 4 μL of 5X Buffer, and 1 μL of M-MLV. Then, incubate at 42°C for 1 hour and at 70°C for 10 minutes to obtain cDNA, which is then stored at -80°C.

[0116] (3) Template amplification

[0117] use PCR was performed using the Hot Start High-Fidelity 2X Master Mix Kit (NEB, M0494S) and an upstream primer (T7-LincGET1-F) and a downstream primer (T7-LincGET1-R) containing the T7 promoter sequence to obtain a 6285-bp LincGET sequence (SEQ ID NO: 1). The LincGET sequence was then purified using the Zymoclean Gel DNA Recovery Kit, measured for concentration, and stored at -20°C for use as a template for LincGET in vitro transcription.

[0118] (4) In vitro transcription and purification

[0119] Using the mMESSAGE mMACHINE_T7 ULTRA kit (Invitrogen, AM1345-5), 10 μL of T72X NTP / ARC, 2 μL of 10X T7 Reaction Buffer, 12 μL of UTP, 10 μg of the LincGET in vitro transcription template obtained in step (3), and an appropriate amount of water were mixed to a final volume of 20 μL. The mixture was incubated at 37°C for 1 hour. 1 μL of TURBO DNase was added to each mixture and incubated at 37°C for 15 minutes. Subsequently, 20 μL of 5X E-PAP Buffer, 10 μL of 25 mM MnCl2, 10 μL of ATP Solution, 4 μL of E-PAP, and 36 μL of water were added and incubated at 37°C for 30 minutes. 200 μL of phenol / chloroform / isoamyl alcohol was added to each 200 μL system, mixed, and centrifuged at 15,000 r at 4°C for 10 minutes. Transfer the supernatant to a new tube, add 1 / 10 of the initial volume of 3MACNa and 2.5 times the volume of ethanol, mix well, and let it stand at -80°C for 1 hour. Then centrifuge at 15,000 r / min at 4°C for 30 minutes. Remove the supernatant and wash three times with 70% ethanol. Dry on ice and dissolve in water to obtain LincGET RNA, which is stored at -80°C.

[0120] 1.2.2 Kdm4d RNA

[0121] use The Kdm4d in vitro transcription template (SEQ ID NO: 3) was amplified from a plasmid containing Kdm4d cDNA (pCMV-T7-kdm4dNM_001410506.1-MTkey, Ubest Biotech) using primers containing the T7 promoter sequence (T7-Kdm4d-F and T7-Kdm4d-R) using a Hot Start High-Fidelity 2X Master Mix kit (NEB, M0494S).

[0122] use Using the T7 ARCA mRNA Kit (NEB, E2060S), 10 μL of ARCA / NTP Mix, 1 μg of Kdm4d in vitro transcription template, 2 μL of T7 RNA Polymerase Mix, and an appropriate amount of water were mixed to a final volume of 20 μL and incubated at 37°C for 30 minutes. 2 μL of DNase I was added to each mixture and incubated at 37°C for 15 minutes. Subsequently, 10 μL of 10X Poly(A) Polymerase Reaction Buffer, 5 μL of Poly(A) Polymerase, and 65 μL of water were added and incubated at 37°C for 30 minutes. 200 μL of phenol / chloroform / isoamyl alcohol was added to each 200 μL volume, mixed, and centrifuged at 15,000 rpm at 4°C for 10 minutes. Transfer the supernatant to a new tube, add 1 / 10 of the initial volume of 3MACNa and 2.5 times the volume of ethanol, mix well, and let it stand at -80°C for 1 hour. Then centrifuge at 15000r at 4°C for 30 minutes. After removing the supernatant, wash three times with 70% ethanol, dry on ice, and dissolve in water to obtain Kdm4d RNA, which is stored at -80°C.

[0123] 1.3 Preparation of ICSI and ROSI Embryos

[0124] 1.3.1 ICSI embryos

[0125] Spermatozoa and round spermatids were obtained from male C57BL / 6 mice (purchased from Beijing Weitonglihua).

[0126] (1) Sperm acquisition

[0127] Remove the male mouse's epididymal tail and place it in M2. Use sterile forceps to express the sperm from the head. Place the expressed sperm in an EP tube containing M2. Keep on ice. Then, use an ultrasonic disruptor to fragment the sperm tail and set aside. After fragmentation, at least 50% of the sperm in the sperm pellet should be motile.

[0128] (2)ICSI procedure

[0129] The inner diameter of the injection needle is about 9-10 μm. The parameters of the piezoelectric membrane rupture instrument are: small pulse less than 3, and the large pulse can be adjusted according to the situation.

[0130] Inject the sperm head into the cytoplasm of the MII stage oocyte obtained in step 1.1, and then culture directly in M16 medium.

[0131] 1.3.2 ROSI embryos

[0132] (1) Take the testicles of 6-8 week old mature male mice and place them in PBS. Then, take 1 / 8 of the testicles and mince them with sterilized scissors. Then add 1 ml of 0.25% trypsin and incubate in a 37°C, 5% CO2 incubator for 8-10 minutes (shaking every 2 minutes).

[0133] (2) Add 2 times the volume of 10G to terminate the digestion. After passing through the cell sieve, centrifuge at 800 rpm for 3 minutes and discard the supernatant.

[0134] (3) Add 2 ml of preheated 10G and 2 μl of Hochest solution, incubate in an incubator for 15 min, and shake once every 5 min.

[0135] (4) Add 5 ml of 10G to terminate the staining, centrifuge at 800 rpm for 3 min, discard the supernatant, and resuspend in 350 μl of 10G.

[0136] (5) After passing through a cell sieve, place the sperm in a 1.5 ml EP tube and sort the haploid spherical sperm using a flow cytometer. Collect the sperm in a 1.5 ml EP tube pre-filled with 200 μl 10G.

[0137] (6) After flow cytometry, centrifuge the liquid at 1500 rpm for 5 minutes, discard the supernatant, and then add 100 μl of M2 culture medium to resuspend. Centrifuge again at 1500 rpm for 5 minutes, discard the supernatant, add M2, and store in a refrigerator at 4°C. 20,000 to 30,000 cells are appropriate.

[0138] (7) The MII stage oocytes obtained in step 1.1 were pre-activated in the incubated strontium chloride activation solution for 10 minutes. They were then removed from the cell culture chamber and the spherical sperm were injected into the oocytes.

[0139] (8) After the operation is completed, activate for 6 hours, remove the cells and culture them in M16 medium, or transplant them.

[0140] ICSI and ROSI procedures were performed in M2 medium (Sigma) containing 5 mg / ml cytochalasin B (Abcam), and all embryos were cultured in a 5% CO 2 incubator at 37°C (Thermo Fisher Scientific).

[0141] 1.4 LincGET and Kdm4d injection into ROSI embryos

[0142] Using an Eppendorf microinjection pipette tip, load approximately 3 μL of the centrifuged material into the injection needle. Use M2 medium as the working medium. Tap the injection needle against the outer wall of an egg-holding needle (90 μm outer diameter, 30 μm inner diameter) to create a small opening. Then adjust the microinjection instrument to an appropriate flow rate (just enough to enlarge the pronucleus without rupturing it).

[0143] Adjust the focus so that the embryo's pronucleus is clearly visible. Gently prick the oocyte with the injection needle until the nuclear membrane is punctured. When the nuclear membrane bulges, quickly withdraw the needle, and the injection is complete. Once all injections are complete, culture the oocyte in KSOM.

[0144] 19 hours after round sperm injection, 2-cell ROSI embryos were used for RNA injection. Using an Eppendorf micromanipulator, a random blastomere was selected and 1-2 μl of 150 ng / μL Kdm4d RNA or 250 ng / μL LincGET RNA obtained in step 1.2 was injected into the nucleus.

[0145] Example 2: ROSI embryo development efficiency detection

[0146] Preparation of ligated male mice

[0147] (1) Select healthy male CD-1 mice aged 6-8 weeks (purchased from Beijing Weitonglihua), anesthetize them with chloral hydrate, disinfect their abdomens with alcohol, and set aside.

[0148] (2) Use sterile surgical forceps and scissors to make an opening of about 0.5 cm in the lower abdomen above the testicles of the male mouse, push the testicles and other reproductive organs out of the abdominal cavity, and use forceps to remove them.

[0149] (3) Heat the forceps until they are red hot, and then find the vas deferens and burn it off. You need to remove about 0.5 cm of the vas deferens.

[0150] (4) Return all reproductive organs to the abdominal cavity, suture the wound, and return the mouse to the mouse room for 3 weeks before use.

[0151] Preparation of pseudo-pregnant mice

[0152] (1) Select estrus mice from the CD-1 mouse population and use the color change and redness and swelling of the vaginal opening as the judgment criteria.

[0153] (2) Select female mice in estrus with engorged vaginal openings and place them in a cage with sterilized male mice. Check the next morning to see if there is a sperm plug. Mice with sperm plugs at the vaginal openings are 0.5-day pseudopregnant mice.

[0154] 2-cell embryo transfer

[0155] Anesthetize the female mouse, pluck the hair from the flank, and disinfect with 75% alcohol. Make an opening on the ventral thigh and remove the oviduct and other organs in the abdominal cavity. Use a mouth pipette with an inner diameter of about 150 μm to select the two-cell embryos (ICSI embryos obtained in Example 1, ROSI embryos injected with LincGET RNA or Kdm4d RNA or water) from the culture medium, transfer them to the egg retrieval solution, and wash off the paraffin oil. Find the oviduct dilatation under the microscope. Use a mouth pipette to inhale three small sections of air, in the middle, and finally inhale a section of air. Use a sterile 1 mL syringe needle to make a small opening on the ovarian side of the dilatation, and at this time, blow the embryo into the dilatation. Place each organ back into the abdominal cavity, suture the muscle sheath, and return to the mouse room after disinfection.

[0156] Mice were delivered by cesarean section 19.5 days after embryo transfer. The number of embryos transferred and the number of mice born were counted according to the different groups of embryos transferred. The birth efficiency of mice in each transplantation group was calculated as follows: birth efficiency = number of mice born / number of embryos transferred. Detailed results are shown in Table 2 below.

[0157] Table 2: Embryo development efficiency

[0158] Group Number of 2-cell embryos transferred (total) Number of mice born (total) Birth efficiency (%) ICSI 60 32 <![CDATA[(53.3±7.6) ab ]]> ROSISB Kdm4d 47 20 <![CDATA[(42.9±9.4) b ]]> ROSISB LincGET 135 54 <![CDATA[(40.2±7.6) b ]]> ROSIDB LincGET 106 25 <![CDATA[(24.1±5.5) cd ]]> ROSI 138 28 <![CDATA[(20.2±4.8) cd ]]> ROSIzygote Kdm4d 59 8 <![CDATA[(14.0±2.7) d ]]> ROSIDB Kdm4d 78 7 <![CDATA[(8.5±2.2) d ]]> ROSIzygote LincGET 60 4 <![CDATA[(6.7±2.9) d ]]>

[0159] SB represents injection into a single blastomere of a two-cell embryo, DB represents injection into both blastomeres of a two-cell embryo, and zygote represents injection into a fertilized egg (zygote). The ICSI group received no injection, and the ROSI group received an injection of water equal to that of the other groups.

[0160] The experimental results showed that injecting LincGET RNA or Kdm4d RNA into a single blastomere of a two-cell embryo, or injecting LincGET RNA into both blastomeres of a two-cell embryo, could effectively improve the birth efficiency of ROSI embryos.

[0161] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings disclosed, and that such modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. The use of item (1) or (2) below in the preparation of a medicament for improving the efficiency of embryonic development in mammals, (1) a nucleic acid molecule encoding LincGET; or (2) Nucleic acid molecules encoding LincGET and nucleic acid molecules encoding Kdm4d.

2. The use according to claim 1, wherein Introducing item (1) or (2) into a mammalian oocyte or embryonic cell; Preferably, the oocyte is an oocyte in metaphase of the second meiotic division; Preferably, the embryonic cell is a two-cell stage embryonic cell; Preferably, the two-cell stage embryonic cell comprises two cells, and item (1) or item (2) is introduced into any one of the two-cell stage embryonic cells, or item (1) or item (2) is introduced into both cells of the two-cell stage embryonic cell; Preferably, item (1) or item (2) is introduced into an oocyte or embryonic cell of a mammal by injection; Preferably, the injection is microinjection.

3. The use according to claim 1 or 2, wherein The embryonic cells are obtained by round spermatid injection (ROSI), intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT).

4. The use according to any one of claims 1 to 3, wherein The drug is administered in the form of a solution; Preferably, the solution comprises a nucleic acid molecule encoding LincGET, and the concentration of the nucleic acid molecule encoding LincGET in the solution is 150-350 ng / μL; Preferably, the concentration of the nucleic acid molecule encoding LincGET in the solution is 200-300 ng / μL; Preferably, the drug further comprises a nucleic acid molecule encoding Kdm4d, and the concentration of the nucleic acid molecule encoding Kdm4d in the solution is 100-200 ng / μL; Preferably, the concentration of the nucleic acid molecule encoding Kdm4d in the solution is 120-180 ng / μL; Preferably, the solution is an aqueous solution.

5. The use according to any one of claims 1 to 4, wherein The nucleic acid molecule encoding LincGET and / or the nucleic acid molecule encoding Kdm4d is introduced into the embryonic cells of a mammal in one of the following forms: isolated nucleic acid molecules (e.g., DNA or RNA), nucleic acid constructs, recombinant expression vectors, liposome particles; Preferably, the nucleic acid molecule encoding LincGET comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the nucleic acid molecule encoding Kdm4d comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

6. The use according to any one of claims 1 to 5, wherein The embryo development efficiency includes blastocyst development efficiency, embryo implantation rate or birth rate.

7. The use according to any one of claims 1 to 6, wherein The mammal is one or more selected from humans, monkeys, gorillas, rats, mice, pigs, cattle, sheep and horses; Preferably, the mammal is a non-human mammal.

8. A method for treating mammalian embryonic cells in vitro, comprising the step of introducing the following item (1) or (2) into the mammalian embryonic cells: (1) a nucleic acid molecule encoding LincGET; or (2) Nucleic acid molecules encoding LincGET and nucleic acid molecules encoding Kdm4d.

9. The method according to claim 8, wherein The embryonic cells are two-cell stage embryonic cells; Preferably, the two-cell stage embryonic cell comprises two cells, and item (1) or item (2) is introduced into any one of the two-cell stage embryonic cells, or item (1) or item (2) is introduced into both cells of the two-cell stage embryonic cell; Preferably, item (1) or item (2) is introduced into embryonic cells of a mammal by injection; Preferably, the injection is microinjection.

10. The method according to claim 8 or 9, wherein The embryonic cells are obtained by round spermatid injection (ROSI), intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT).

11. The method according to any one of claims 8 to 10, wherein Item (1) or (2) is administered in the form of a solution; Preferably, the concentration of the nucleic acid molecule encoding LincGET in the solution is 150-350 ng / μL; Preferably, the concentration of the nucleic acid molecule encoding LincGET in the solution is 200-300 ng / μL; Preferably, the concentration of the nucleic acid molecule encoding Kdm4d in the solution is 100-200 ng / μL; Preferably, the concentration of the nucleic acid molecule encoding Kdm4d in the solution is 120-180 ng / μL.

12. The method according to any one of claims 8 to 11, wherein The nucleic acid molecule encoding LincGET and / or the nucleic acid molecule encoding Kdm4d is introduced into the embryonic cells of a mammal in one of the following forms: isolated nucleic acid molecules (e.g., DNA or RNA), nucleic acid constructs, recombinant expression vectors, liposome particles; Preferably, the nucleic acid molecule encoding LincGET comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the nucleic acid molecule encoding Kdm4d comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

13. The method according to any one of claims 8 to 12, wherein The mammal is one or more selected from humans, monkeys, gorillas, rats, mice, pigs, cattle, sheep and horses; Preferably, the mammal is a non-human mammal.

14. An assisted reproductive method comprising the following steps: The method for treating mammalian embryonic cells in vitro according to any one of claims 8 to 13, wherein the treated mammalian embryonic cells are obtained.

15. The assisted reproductive method according to claim 14, wherein: The method further comprises the step of transplanting the embryonic cell into a subject; Preferably, the embryonic cells are transplanted at the two-cell stage; Preferably, the mammal and the subject are the same individual or different individuals of the same species; Preferably, the mammal and the subject are non-human mammals.

16. The assisted reproductive method according to claim 14 or 15, wherein: The assisted reproductive method is for non-therapeutic purposes.

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