阅读说明：本技术 一种组合物及其应用 (Composition and application thereof ) 是由 吴光明 陈捷凯 于 2020-05-22 设计创作，主要内容包括：本发明属于生物医药领域,涉及一种组合物及其应用。本发明提供了一种组合物,所述组合物成分包括甘露醇、MgSO<Sub>4</Sub>、CaCl<Sub>2</Sub>和牛血清白蛋白；所述甘露醇、MgSO<Sub>4</Sub>、CaCl<Sub>2</Sub>和牛血清白蛋白的质量比为9～53：0.015～0.241：0.013～0.23：0.01～5。所述的组合物作为电融合液用于细胞融合,能够使得融合效率提高到100％,用于制备基因编辑小鼠,能够明显促进基因编辑小鼠的存活率。(The invention belongs to the field of biological medicines, and relates to a composition and application thereof. The invention provides a composition, and the components of the composition comprise mannitol and MgSO 4 、CaCl 2 And bovine serum albumin; the mannitol and MgSO 4 、CaCl 2 And bovine serum albumin in a mass ratio of 9-53: 0.015 to 0.241: 0.013-0.23: 0.01 to 5. The composition is used as an electrofusion liquid for cell fusion, can improve the fusion efficiency to 100 percent, is used for preparing a gene editing mouse, and can obviously promote the survival rate of the gene editing mouse.)

1. A composition comprising mannitol, MgSO₄、CaCl₂And bovine serum albumin; the mannitol and MgSO₄、CaCl₂And bovine serum albumin in a mass ratio of 9-53: 0.015 to 0.241: 0.013-0.23: 0.01 to 5.

2. The composition of claim 1, wherein the mannitol, MgSO₄、CaCl₂And bovine serum albumin in a mass ratio of 18-52: 0.018-0.241: 0.016-0.23: 0.01-4;

preferably, the mannitol, MgSO₄、CaCl₂And bovine serum albumin at a mass ratio of 27-52: 0.018-0.12: 0.016-0.1: 1 to 3.5;

or preferably, the composition comprises the following ingredients: mannitol 0.05-0.29M, MgSO₄0.12-2mM、CaCl₂0.12-2mM, bovine serum albumin 0.01-5 mg/mL;

more preferably, the compositionComprises the following components: mannitol 0.1-0.28M, MgSO₄0.15-2mM、CaCl₂0.15-2mM, bovine serum albumin 0.01-4 mg/mL;

more preferably, the composition comprises the following ingredients: mannitol 0.15-0.28M, MgSO₄0.15-1mM、CaCl₂0.15-1mM, bovine serum albumin 1-3.5 mg/mL;

more preferably, the composition comprises the following ingredients: mannitol 0.18-0.28M, MgSO₄0.15-0.5mM、CaCl₂0.15-0.5mM, bovine serum albumin 2-3.5 mg/mL;

even more preferably, the composition comprises the following ingredients: mannitol 0.2-0.28M, MgSO₄0.15-0.3mM、CaCl₂0.15-0.3mM and 2-3mg/mL bovine serum albumin.

Or preferably, the composition is an electrofusion fluid;

more preferably, the composition is used to prepare an animal;

more preferably, the animal is a gene-editing animal embryonic cell or a gene-editing animal;

more preferably, the animal is a genetically humanized animal;

more preferably, the composition is used to prepare animal tetraploid embryos;

more preferably, the animal is a mammal;

further preferably, the mammal is a rodent;

further preferably, the rodent is a mouse.

3. Use of a composition according to any one of claims 1-2 for the preparation of an animal embryonic cell or animal;

preferably, the embryonic cell is a gene-editing animal embryonic cell;

or preferably, the animal is a gene-editing animal;

more preferably, the animal is a genetically humanized animal;

or preferably, the composition is used to prepare animal tetraploid embryos;

more preferably, the animal is a mammal;

further preferably, the mammal is a rodent;

further preferably, the rodent is a mouse.

4. An electrofusion liquid containing mannitol, bovine serum albumin, and Mg²⁺And Ca²⁺Characterized in that the electrofusion fluid has an increased concentration of Mg²⁺And Ca²⁺(ii) a Or, the electrofusion liquid Mg²⁺And Ca²⁺The concentration of (A) is 0.12-2mM, 0.12-2mM respectively;

preferably, the concentrations of mannitol and bovine serum albumin in the electrofusion liquid are 0.05-0.29M and 0.01-5mg/mL respectively;

more preferably, said Mg²⁺And Ca²⁺Respectively derived from MgSO₄And CaCl₂。

5. A method for producing an animal, characterized in that the electrofusion liquid of claim 4 is used;

preferably, the animal is a gene-editing animal;

more preferably, the animal is a genetically humanized animal.

More preferably, the composition is used to prepare animal tetraploid embryos;

more preferably, the animal is a mammal;

more preferably, the mammal is a rodent;

more preferably, the rodent is a mouse;

more preferably, the animal is an adult mouse;

or more preferably, the animal is a fetal mouse.

6. The method of claim 5, wherein the method uses a tetraploid compensation method or a tetraploid embryo complementation method;

preferably, the method comprises polymerizing the embryonic stem cells and tetraploid embryos to form new reconstituted embryos.

7. The method of claim 5, wherein the method comprises the steps of:

(1) obtaining a mouse 2-cell embryo;

(2) putting the 2-cell embryo in the step (1) into an electric fusion liquid for electric fusion to obtain a tetraploid embryo;

(3) putting the tetraploid embryos obtained in the step (2) into a culture medium for culturing;

(4) polymerizing the tetraploid embryos and the embryonic stem cells in the step (3) to form chimeric embryos;

(5) implanting the chimeric embryo in the step (4) into the uterus of a pseudopregnant mouse, and developing the embryo to a foot pregnancy so as to obtain a gene-edited mouse;

preferably, the culturing time in step (3) is 8-24 hours;

or preferably, the medium in step (3) is KSOM medium.

8. A method for producing a tetraploid embryo or a chimera embryo, wherein the composition of any one of claims 1-2 is used;

preferably, the composition is as an electrofusion fluid;

more preferably, the composition is used to prepare animal tetraploid embryos;

more preferably, the composition is used to prepare chimeric embryos;

more preferably, the method comprises polymerizing the tetraploid embryo with an embryonic stem cell to obtain a chimeric embryo.

9. A mouse produced by the method of any one of claims 5-7.

10. A gene-editing mouse or the tissue, body fluid, cell, nucleus of its offspring, or disrupted fragments or extracts thereof, wherein the mouse is constructed by the method according to any one of claims 5 to 7.

Technical Field

The invention belongs to the field of biological medicines, and relates to a composition and application thereof.

Background

Tetraploids refer to biological individuals or cells that contain four times the minimum number of chromosomes in the cell, i.e., cells having four complete chromosomes in the cell. Under natural conditions, mammalian tetraploid embryos are extremely low in incidence and are not commonly capable of developing normally into a single individual.

A certain number of Embryonic Stem (ES) cells and tetraploid embryos are chimeric, and the ES cells and tetraploid embryos are not randomly distributed during the development of the chimeras, i.e., the tetraploid embryo parts are only involved in the formation of extraembryonic tissues such as yolk sac endoderm and placental trophoblast cells (such as chorioectoderm, trophoblast cells, etc.), while the ES cells are widely involved in the formation of parts of embryo bodies, allantois, amnion, yolk sac mesoderm and chorion mesoderm, but not in the formation of cell lineages of yolk sac endoderm and placental trophoblast, i.e., the developmental potentials of the two have complementarity, which is called tetraploid complementation technology.

The individual animals cloned by this technique, which develop entirely from ES cells, are called ES animals. The birth rate of the fetus after the cloned embryo prepared by the cell nucleus transplantation method is transplanted to a surrogate mother is very low and is only 1-2%, and the birth rate of the fetus can be improved to 20-30% by injecting an ESC cell line into a tetraploid embryo carrier in a blastocyst stage through a tetraploid compensation technology, so that the animal cloning efficiency is obviously improved. In recent years, many scientists at home and abroad deeply research the technology and obtain a plurality of creative research results. Tetraploid compensation techniques produce animal models with very general applications such as determining lineage specific gene function, analyzing extraembryonic and embryonic gene function, identifying cellular spontaneous and non-spontaneous gene function, distinguishing major and minor defects, facilitating phenotypic analysis, etc. The application of the embryo chimera is not limited to the research of early embryo development, but also can be applied to the research of postnatal fetal development and physical function. When chimeras are used in conjunction with molecular tools, such molecular tools can modify genetic activity in cells in a time and lineage specific manner, facilitating accurate, in-depth, and large-scale analysis of gene function. Genetic activity (Nagy 2000) or regulation of gene function can be altered by means of conditionally altering gene expression. The current evaluation of the developmental potential of ES cells has focused mainly on studying cell differentiation in vitro, and on the analysis of transcriptional activity reflecting cellular pluripotency molecular markers. One unique feature of embryo chimerism is that foreign cells can be chimerized into the embryo, and the cells can fully stimulate differentiation potential during embryonic development. Cells were tested for a comprehensive cell lineage, which more reveals the true extent of pluripotency. Chimeras are the most comprehensive and most stringent of important tools for predicting mammalian pluripotency cellular in vivo evaluations.

Disclosure of Invention

The invention provides a preparation method of animals.

The invention provides an electrofusion liquid.

The invention provides a preparation method capable of improving the birth rate of animals.

The invention provides a tetraploid compensation or complementation method (tetraploid organization) for animals.

In some embodiments, a composition is used in the method of making an animal, the composition comprising mannitol, MgSO₄、CaCl₂And bovine serum albumin; the mannitol and MgSO₄、CaCl₂And bovine serum albumin in a mass ratio of 9-53: 0.015 to 0.241: 0.013-0.23: 0.01 to 5.

In some embodiments, the method uses a composition comprising mannitol, MgSO₄、CaCl₂And bovine serum albumin; the mannitol and MgSO₄、CaCl₂And bovine serum albumin in a mass ratio of 9-53: 0.015 to 0.241: 0.013-0.23: 0.01 to 5.

In some embodiments, mannitol, MgSO, in the composition₄、CaCl₂And bovine serum albumin in a mass ratio of 18-52: 0.018-0.241：0.016～0.23：0.01-4。

In some embodiments, mannitol, MgSO, in the composition₄、CaCl₂And bovine serum albumin at a mass ratio of 27-52: 0.018-0.12: 0.016-0.1: 1 to 3.5.

In some embodiments, the composition comprises the following ingredients: mannitol 0.05-0.29M, MgSO₄0.12-2mM、CaCl₂0.12-2mM, bovine serum albumin 0.01-5 mg/mL.

In some embodiments, the composition comprises the following ingredients: mannitol 0.1-0.28M, MgSO₄0.15-2mM、CaCl₂0.15-2mM, bovine serum albumin 0.01-4 mg/mL.

In some embodiments, the composition comprises the following ingredients: mannitol 0.15-0.28M, MgSO₄0.15-1mM、CaCl₂0.15-1mM, bovine serum albumin 1-3.5 mg/mL.

In some embodiments, the composition comprises the following ingredients: mannitol 0.18-0.28M, MgSO₄0.15-0.5mM、CaCl₂0.15-0.5mM, bovine serum albumin 2-3.5 mg/mL.

In some embodiments, the composition comprises the following ingredients: mannitol 0.2-0.28M, MgSO₄0.15-0.3mM、CaCl₂0.15-0.3mM and 2-3mg/mL bovine serum albumin.

In some embodiments, the composition comprises the following ingredients: mannitol 0.27M, MgSO₄0.2mM、CaCl₂0.2mM, bovine serum albumin 3 mg/mL.

In some embodiments, the composition may be used as a mother liquor and then formulated into a working fluid of desired concentration.

In some embodiments, the composition may be a working fluid and may be used without formulation.

In some embodiments, the invention provides for the use of the composition in the preparation of a gene-edited animal embryonic cell or a gene-edited animal.

In some embodiments, the composition is used to prepare animal tetraploid embryos.

In some embodiments, the method uses a methodAn electrofusion liquid containing mannitol, bovine serum albumin and Mg²⁺And Ca²⁺The electrofusion fluid has an increased concentration of Mg²⁺And Ca²⁺(ii) a Or, the electrofusion liquid Mg²⁺And Ca^{2 +}The concentration of (B) is 0.12-2mM, respectively.

In some embodiments, the concentrations of mannitol and bovine serum albumin in the electrofusion fluid are 0.05-0.29M and 0.01-5mg/mL, respectively.

In some embodiments, the Mg²⁺And Ca²⁺Respectively derived from MgSO₄And CaCl₂。

In some embodiments, the electrofusion fluid is used to prepare tetraploid embryos.

At present, tetraploids are obtained by biological, chemical and physical methods. Injecting two diploid embryonic cell nucleuses into cytoplasm of 1 embryonic cell at 1-cell stage by micromanipulation, which has high requirements on operation technology, and meanwhile, the micromanipulation has great damage to embryos; the use of cytochalasin B or colchicines to inhibit the division of blastomeres to prepare tetraploid can cause adverse effects on embryonic development and even delay the retardation of embryonic development; in addition, the method of Sendai virus, polyethylene glycol, DC electric shock and the like can be used for cell fusion, the Sendai virus has pathogenicity and residual toxicity of polyethylene glycol, and the electric fusion method can only generate reversible electroporation in a short time, overcomes the toxicity of chemical reagents and the pathogenicity of viruses, and is the most common and efficient method for preparing tetraploid embryos. However, the existing tetraploid compensation technology has the problems that the embryo electrofusion efficiency is not 100 percent, the efficiency and the stability of mouse preparation are poor, and the efficiency of embryonic stem cells from a pure mouse is low. During the research process, the inventor unexpectedly finds that the fusion efficiency of the improved electric fusion liquid can be improved from 80-90% to 100%, and has great practical significance.

In some embodiments, the present invention provides a method of making an animal comprising polymerizing a tetraploid embryo with an embryonic stem cell to form a new reconstituted or chimeric embryo; the tetraploid embryo is in 2-cell stage.

In some embodiments, the embryonic stem cells refer to embryonic stem cells with passage numbers within p 15.

In some embodiments, the embryonic stem cells refer to embryonic stem cells at passage numbers p12-p 15.

In some embodiments, the methods use tetraploid compensation or tetraploid embryo complementation.

In some embodiments, the method comprises the steps of: (1) obtaining an animal 2-cell embryo; (2) putting the 2-cell embryo in the step (1) into an electric fusion liquid for electric fusion to obtain a tetraploid embryo; (3) putting the tetraploid embryos obtained in the step (2) into a culture medium for culturing; (4) polymerizing the tetraploid embryos developed to the 2-cell stage in the step (3) with embryonic stem cells to form chimeric embryos; (5) implanting the chimeric embryo in the step (4) into the uterus of a pseudopregnant animal, and developing the embryo to a foot pregnancy so as to obtain the animal.

In some embodiments, the incubation time in step (3) is 8-24 hours.

In some embodiments, the medium in step (3) is KSOM medium.

In some embodiments, the non-human animal derived from the recipient embryo can be any animal other than a human, such as a pig, rat, mouse, hamster, rabbit, pig, cow, deer, sheep, goat, chicken, cat, horse, dog, orangutan, monkey. Meanwhile, the mammal used as the original source of the cells transplanted into the recipient may be a human or a mammal other than a human, for example, strain, rat, mouse, cow, sheep, goat, horse, dog, baboon, chimpanzee, gorilla, orangutan, monkey, marmoset, and bonobo, etc.

In some embodiments, the animal is a mammal.

In some embodiments, the animal is a non-human mammal.

In some embodiments, the animal is selected from the group consisting of a pig, rat, mouse, hamster, rabbit, pig, cow, deer, sheep, goat, chicken, cat, horse, dog, chimpanzee, monkey.

In some embodiments, the animal is selected from a mouse. In some embodiments, the animal is selected from adult mice. In some embodiments, the animal is selected from a fetal rat.

Drawings

FIG. 1 is a flowchart and a result chart of the mouse preparation in example 2.

FIG. 2 is a flowchart showing the procedure and results of the preparation of mice in test example 1.

Fig. 3 is a graph showing the results of mouse birth rates.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same definitions as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials being described in the detailed description.

As used herein, "a" and "an" refer to the definitions of grammatical indefinite articles, meaning "a", "an" or "a plurality" of "or" a plurality "(i.e.," at least one "). For example, "an element" means one or more of the elements.

In this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements; i.e. open definition.

Herein, the term "tetraploid complementation technique" is equivalent to "tetraploid embryo compensation technique" or "tetraploid complementation method" or "tetraploid embryo complementation method", which means that a certain number of Embryonic Stem (ES) cells and tetraploid embryos are chimeric, and during the development of the chimera, the ES cells and tetraploid embryos are not randomly distributed, i.e., the tetraploid embryo portion is involved in the formation of extraembryonic tissues such as yolk sac endoderm and placental trophoblast cells (e.g., chorioectoderm, trophoblast cells, etc.), while the ES cells are widely involved in the formation of embryonic somatic, allantoic, amniotic, yolk sac mesoderm and chorion mesoderm portions, and not in the formation of yolk sac endoderm and placental trophoblast cell lineages, i.e., the developmental potentials of the two are complementary, which is called tetraploid complementation technique.

In some instances, the term "embryo" refers to tissues and cells derived from or within an animal subject at any time prior to birth.

In some cases, "embryo" is used to describe a fertilized oocyte that becomes a pre-fetal movement after implantation into the uterus eight weeks after fertilization. According to this definition, a fertilized oocyte is commonly referred to as a pre-embryo (pre-embryo) prior to transplantation. However, throughout this patent application we will use the broader term embryo definition, which includes the pre-embryonic stage. It therefore includes all stages of development from oocyte fertilization to morula, hatching and implantation during the blastocyst stage.

The embryo is approximately spherical and consists of one or more cells (blastomeres) surrounded by a gelatin-like shell, and the acellular matrix is called zona pellucida. The zona pellucida performs multiple functions until the embryo hatches, and is a good marker for embryo evaluation. The band is spherical and translucent and should be clearly distinguishable from cellular debris.

The mammal embryo before implantation has a diameter of about 90-120 microns and is coated with a clear and visible zona pellucida composed of glycoprotein. On one side of the fertilized egg, 1 to 2 polar bodies formed at the time of meiosis were observed.

An embryo with 2 blastomeres formed after the first mitosis of the ovum between 23 and 43.5 hours after fertilization. The cells of the embryo at the 2-cell stage are nearly elliptical and are basically equal in size, and 2 blastomeres can be fused into a 4-ploid cell after electric shock, so that the 4-ploid embryo is developed. The fertilized egg is divided into two to form 2 blastomeres, namely a 2-cell stage, and further, the 2 blastomeres are divided into two to form 4 blastomeres, namely a 4-cell stage; and the number of the morula is 8, namely the 8-cell stage, and the morula is formed when the morula is divided into 32 cells after 5 times. At the 4-cell stage, 4 blastomeres were seen in a staggered arrangement. The 8-cell stage blastomere arrangement is divided into upper and lower layers, and densification begins to occur with the formation of tight junctions. The 16-cell phase begins with the appearance of inner cell mass and trophoblast differentiation, called morula. After fertilization, the embryo develops to the 32-cell stage 72 hours, gaps appear among blastomeres, a complete blastocyst cavity is formed after the cracks are enlarged, and one side of the cavity is concentrated with a group of cells, namely an inner cell mass. A layer of flat cells, called trophoblasts, surrounds the periphery of the blastocoel. The blastocyst is also referred to herein as a blastocyst. The blastocyst expands at the late stage and the zona pellucida is extruded, thus the hatching blastocyst is obtained.

The term embryo is used to denote the various stages-fertilized oocyte, fertilized egg (zygate), 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, expanded blastocyst and hatched blastocyst, and all stages in between (e.g., 3-cell or 5-cell).

The term "chimeric blastocyst" or "chimeric embryo" as used herein refers to a blastocyst or embryo comprising an embryonic stem cell in a chimeric state. The chimeric blastocysts or embryos can be produced, in addition to by injection methods, using, for example, the so-called "aggregation method" in which embryos + embryos, or embryo + cells, are caused to adhere tightly to one another in a petri dish to produce chimeric blastocysts. Further, the relationship between the recipient blastocyst or embryo and the cell to be transplanted in the present disclosure may be an allogeneic or xenogeneic relationship.

The term "animal" in reference to a donor cell and/or host embryo includes mammals, fish and birds. Mammals include, for example, humans, non-human primates, rodents (e.g., mice, rats, hamsters, guinea pigs), livestock (e.g., bovine species, e.g., cows, steers, etc.; ovine species, e.g., sheep, goats, etc.; and porcine species, e.g., pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, and the like. The phrase "non-human mammal" referring to a donor cell and/or host embryo excludes humans.

In various embodiments, the donor cell and/or host embryo are not from one or more of the following: protomurine species (Akodon spp.), Trautuma species (Myopus spp.), volvulus species (Microtus spp.), Scaptochirus species (Talpa spp.).

In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from the superfamily murinus. In one embodiment, the genetically modified animal is from a family selected from the following families: poppy (calomycidae) (e.g., cricetulus), Cricetidae (Cricetidae) (e.g., hamster, new world rat, vole), Muridae (Muridae) (true rat, gerbil, spiny rat, corolla), aestividae (nesomyidae) (climbing rat, rock climbing rat, white tail rat, equina rat), platathromonidae (platathomyidae) (e.g., spiny rat), and Spalacidae (Spalacidae) (e.g., marsupium rat, bamboo rat, and phenol rat); and the ADAM6 ortholog or homolog is selected from a different species within the same family. In a specific embodiment, the genetically modified rodent is selected from a genuine mouse (muridae) and the ADAM6 ortholog or homolog is from a species selected from a gerbil, a spiny mouse, or a crow mouse. In one embodiment, the genetically modified mouse is a member from the murine family and the ADAM6 ortholog or homolog is from a different species from the murine family. In a specific embodiment, the genetically modified rodent is a mouse of the family muridae, and the ADAM6 ortholog or homolog is from a rat, gerbil, or spiny hair of the family muridae.

Methods for making non-human animals, such as mice, from donor ES cells and host embryos are known in the art. Donor ES cells are selected for specific characteristics that enhance the ability of the cell to propagate the host embryo, and thereby contribute in part or in large part to the animal formed by the donor ES cell and the host embryo. The animals formed may be male or female, most based on the genotype of the ES cell (e.g., XY or XX).

In some embodiments, the cell is a pluripotent cell, a non-pluripotent cell, a mammalian cell, a human cell, a non-human mammalian cell, a rodent cell, a mouse cell, a hamster cell, a non-human pluripotent cell, a rodent pluripotent cell, or a fibroblast or lung cell.

In some of the above methods, the cell is a primary cell or an immortalized cell. In some of the above methods, the rodent pluripotent cells are mouse or rat Embryonic Stem (ES) cells.

In some of the above methods, the animal cell or the human cell is a primary cell or an immortalized cell.

In some of the above methods, the animal cell or the human cell is a pluripotent cell. In some of the above methods, the animal pluripotent cells are mouse Embryonic Stem (ES) cells. In some of the above methods, the human pluripotent cell is a human Embryonic Stem (ES) cell, a human adult stem cell, a developmentally restricted human progenitor cell, or a human Induced Pluripotent Stem (iPS) cell.

In some embodiments, certain embodiments herein provide humanized gene-edited cells, particularly also isolated human and non-human totipotent or pluripotent stem cells, particularly mouse embryonic stem cells, capable of maintaining pluripotency following one or more in vitro continuous genetic modifications and capable of transmitting the targeted genetic modification to progeny via germline.

The term "embryonic stem cell" or "ES cell" as used herein includes any embryo-derived totipotent or pluripotent cell capable of promoting the development of an embryo upon introduction into the embryo. The term "pluripotent cell" as used herein includes undifferentiated cells that have the ability to develop into more than one type of differentiated cell. The term "non-pluripotent cell" includes cells that are not pluripotent cells.

The terms "administration" or "transplantation," as used herein, refer to the placement of a cell in a subject by a method or route that results in the desired effect by at least partially localizing the cell to a desired site.