CN113403259A - Additive for improving development quality of cloned embryos and application thereof - Google Patents
Additive for improving development quality of cloned embryos and application thereof Download PDFInfo
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- CN113403259A CN113403259A CN202110626164.5A CN202110626164A CN113403259A CN 113403259 A CN113403259 A CN 113403259A CN 202110626164 A CN202110626164 A CN 202110626164A CN 113403259 A CN113403259 A CN 113403259A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0273—Cloned vertebrates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2317—Interleukin-17 (IL-17)
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Abstract
The invention discloses an additive for improving the development quality of cloned embryos, which is used for inhibiting apoptosis of embryo cells by adding IL17D into an in vitro cloned embryo culture solution so as to improve the development quality of cloned embryos, is applied to the fields of improving the development quality of cloned embryos, improving the technical efficiency of animal cloning, inhibiting apoptosis of in vitro cultured embryo cells and the like, and is further widely applied to the fields of livestock production, gene editing research and the like.
Description
Technical Field
The invention relates to the technical field of somatic cell cloning, in particular to an additive for improving the development quality of cloned embryos and application thereof.
Background
The Somatic Cell Nuclear Transfer (SCNT) technology, also known as cloning technology, is widely used in The fields of animal production, gene editing research, therapeutic cloning, and The like. In animal production, the cloning technology can be applied to breeding of good and good breeding stock, production of bioreactors, grain-saving, environmental-friendly and disease-resistant variety cultivation. However, the low developmental efficiency of SCNT embryos has been a major reason limiting their expansion. Among the many reasons that result in the low development efficiency of SCNT embryos, the development arrest caused by early embryonic apoptosis has been an important cause. Apoptosis of embryonic cells is beneficial in removing dysplastic cells, but apoptosis beyond a certain extent can lead to embryonic development arrest. Numerous studies have shown that excessive apoptosis, shrinkage of apoptotic cells, chromatin condensation, and DNA fragmentation are observed in pre-spiked embryos. The causes of the SCNT embryonic cell excessive apoptosis include abnormal zygote activation, abnormal chromosome segregation, oocytes, in vitro operation procedures and culture environment. It has been shown that the addition of cell factor IGF1, hormone melatonin and vitamin C can inhibit apoptosis of cloned embryo cell and raise the development efficiency of cloned embryo.
Interleukin 17 (IL 17) is a cytokine family, and 6 members such as IL-17A-F are identified and cloned by genome sequencing and proteomics. The existing research shows that the IL17 family protein mainly plays a role in immunity, enhances the cellular immune response mainly by inducing cells to express proinflammatory cytokines, chemokines and antibacterial peptides, participates in inflammation and mucosal host defense, and has a certain function in pathogenic bacteria infection and tumorigenesis.
Disclosure of Invention
The invention aims to provide an additive for improving the development quality of cloned embryos and application thereof in the fields of improving the development quality of cloned embryos, improving the cloning efficiency of animals and inhibiting apoptosis of embryo cells cultured in vitro.
In one aspect of the invention, an additive for improving the development quality of cloned embryos is provided, the additive comprising one or more cytokines of the IL17 family.
In certain embodiments, the additive that enhances the quality of development of a cloned embryo is IL 17D.
In certain embodiments, the additive that enhances the quality of development of cloned embryos, IL17D, is present at a concentration of 50-100 ng/mL.
In certain embodiments, the additive that enhances the quality of development of cloned embryos, IL17D, is at a concentration of 50 ng/mL.
In certain embodiments, the additive that improves the quality of development of a cloned embryo, IL17D, is added 0h after activation of the cloned embryo.
The invention also provides application of the additive for improving the development quality of the cloned embryos in improving the development quality of the cloned embryos. The additive is one or more cytokines in IL17 family; preferably, the additive is IL 17D; preferably, the additive is IL17D at a concentration of 50-100 ng/mL; preferably, the additive is IL17D at a concentration of 50 ng/mL; preferably, the additive is IL17D at a concentration of 50ng/mL and is added at 0h after activation of the cloned embryos.
In a third aspect of the invention, the application of the additive for improving the development quality of cloned embryos in the field of improving the animal cloning efficiency is provided. The additive is one or more cytokines in IL17 family; preferably, the additive is IL 17D; preferably, the additive is IL17D at a concentration of 50-100 ng/mL; preferably, the additive is IL17D at a concentration of 50 ng/mL; preferably, the additive is IL17D at a concentration of 50ng/mL and is added at 0h after activation of the cloned embryos.
In a fourth aspect, the invention provides an application of an additive for improving the development quality of cloned embryos in inhibiting apoptosis of in vitro cultured embryo cells. The additive is one or more cytokines in IL17 family; preferably, the additive is IL 17D; preferably, the additive is IL17D at a concentration of 50-100 ng/mL; preferably, the additive is IL17D at a concentration of 50 ng/mL; preferably, the additive is IL17D at a concentration of 50ng/mL and is added at 0h after activation of the cloned embryos.
In a fifth aspect of the present invention, there is provided a method for improving animal cloning efficiency by using an additive for improving the development quality of cloned embryos, wherein the method comprises: preparing fusion activated cloned and reconstructed embryos; culturing the cloned and reconstructed embryos 0h after activation in PZM3 basal medium containing 50ng/mL IL 17D; placing the cloned reconstructed embryo cultured and developed to 2 cell stage into the uterus of surrogate pregnant animal; the cloned animal is bred by inoculation, and the birth rate and healthy litter size of the cloned animal are improved. The additive is one or more cytokines in IL17 family; preferably, the additive is IL 17D; preferably, the additive is IL17D at a concentration of 50-100 ng/mL; preferably, the additive is IL17D at a concentration of 50 ng/mL; preferably, the additive is IL17D at a concentration of 50ng/mL and is added at 0h after activation of the cloned embryos.
In certain embodiments, the methods of increasing the efficiency of animal cloning using additives that increase the quality of cloned embryo development are applicable to pigs, cattle, sheep, mice, or other mammals.
The invention has the beneficial effects that: IL17D is added into the in vitro cloned embryo culture solution to inhibit the apoptosis of embryo cells, so as to improve the development quality of cloned embryos, and the method is applied to the fields of improving the development quality of cloned embryos, improving the technical efficiency of animal cloning, inhibiting the apoptosis of in vitro cultured embryo cells and the like, and further serves the fields of animal husbandry production, gene editing research and the like.
Drawings
FIG. 1 shows the results of Hoechst33342 staining and apoptosis assay (TdT-sized dUTP Nick-End Labeling, TUNEL) of cloned embryos.
FIG. 2 shows the result of apoptosis of cloned embryos treated with IL 17D.
FIG. 3 shows the relative expression level of part of the apoptotic genes in cloned embryos treated with IL 17D.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
1. Construction of cloned pig embryos
Oocyte maturation: preserving pig ovaries in preheated physiological saline containing penicillin and streptomycin sulfate at 37 ℃, selecting follicles with the diameter of about 5mm by using an 18-gauge needle and a syringe, extracting follicular fluid, naturally settling the follicular fluid at 38.5 ℃ for 10min, removing supernatant, adding DPBS (DPBS) for resuspension, repeating the step for 2 times, taking the resuspended fluid, placing the resuspended fluid in a 10cm culture plate, observing under a stereoscope, selecting appropriate Cumulus cell-oocyte complexes (COCs) by using a suction tube, washing the selected COCs for 3 times by using a mature culture medium, transferring the COCs into the in vitro mature culture medium which is balanced for 4 hours, and covering the COCs by using embryo-grade mineral oil. The in vitro maturation medium components were as follows: basal medium 199(Gibco), 1G/L polyvinyl alcohol, 3.05mM glucose, 0.91mM sodium pyruvate, 0.57nM cysteine, 0.5. mu.g/mL luteinizing hormone, 0.5. mu.g/mL follicle stimulating hormone, 10ng/mL epidermal growth factor, 100mL/L pig follicular fluid, 75. mu.g/mL penicillin (penicillin G), and 50. mu.g/mL streptomycin. COCs at 38.5 ℃ and 5% CO2Culturing for 42-44h in the incubator. The cultured COCs were placed in a 1.5mL centrifuge tube containing DPBS containing 1mg/mL hyaluronidase, and gently blown up with a pipette gun to remove cumulus cells from the COCs. Finally, the cells are selectedOocytes with uniform quality, round and smooth cell morphology, clear egg space and clear polar body are used as materials for nuclear transfer experiments.
Donor cell preparation: separating fetal fibroblast from 35 days pig fetus, freezing the separated cells in liquid nitrogen, recovering fetal fibroblast from liquid nitrogen tank 2-3 days before nuclear transplantation, culturing in 12% FBS culture medium at 38.5 deg.C and 5% CO2To a confluency of 90%. Digested with trypsin and resuspended in micromanipulation solution for use.
Activation of cloned and reconstructed embryo fusion: the oocytes were enucleated by blind aspiration and the first polar body and surrounding tissues were removed. And (3) selecting donor cells with better morphology under a microscope, injecting the cell nucleuses of the donor cells into the yolk of the enucleated oocytes from a primary incision, and obtaining cloned and reconstructed embryos. Cloning of reconstructed embryos culturing in porcine zygate medium-3(PZM3) medium at 38.5 ℃ with 5% CO2Incubation for 4h was followed by fusion activation. The PZM3 culture solution comprises the following components: 108.00mM NaCl, 10.00mM KCl, KH2PO40.35 mM、MgSO4·7H2O 0.40mM、NaHCO325.07 mM, sodium Pyruvate (Na-Pyruvate)0.20mM, calcium lactate 2.00mM, taurine (Hypotaurin) 5.00mM, L-glutamine 1.00mM, essential amino acid 20mL/L, and non-essential amino acid 10 mL/L. . Setting the parameters of the electrofusion instrument as 150v/mm, 100 mus and 2DC, adopting fusion-activation liquid to clean the electrofusion groove, adding 500 muL of fusion activation liquid, transferring 10 cloned and reconstructed embryos into the electrofusion groove, rotating by a glass fine needle to ensure that the axis of an egg receptor-nuclear donor is vertical to an electrode, and carrying out fusion activation on the cloned and reconstructed embryos.
2. In vitro culture of porcine cloned embryos and analysis of results of cloning efficiency
2.1 treatment of in vitro Medium PZM3
To the PZM3 basic culture medium, 0ng/mL, 50ng/mL, 100ng/mL IL17D protein was added, and the mixture was incubated for 44h for use.
2.2 Effect of 0h (0 hr) supplementation with IL17D on cloned embryo development after fusion activation
At 0h after activation of cloned embryos, they were placed in plates containing in vitro medium PZM3 supplemented with IL 17D. The number of cleavage, 4 cells and blastula were recorded when the cloned embryos were cultured for 48h, 64h and 168 h. The blastocysts were collected, stained with DPBS containing Hoechst33342 at a concentration of 10. mu.g/ml for 10min, and the number of cells in the blastocysts was observed under a fluorescent microscope. As shown in Table 1, the development efficiency of cloned embryos cultured in-vitro culture medium PZM3 added with IL17D at a concentration of 50ng/mL or 100ng/mL is improved compared with that of a control group (IL 17D at a concentration of 0 ng/mL), wherein the early development efficiency of cloned embryos cultured in a 50ng/mL IL17D treatment group is the highest, the 4-cell rate is improved by 37.7% and the blastocyst rate is improved by 66.2% compared with that of the control group.
TABLE 1 Effect of different concentrations of IL17D on the efficiency of cloned embryo development
2.3 Effect of IL17D supplementation on clonal embryo development at various time periods following embryo activation.
According to the results in 2.2, the 50ng/mL IL 17D-treated group was the optimal concentration group. Thus, after embryo activation, 0h (0 h), 24h (24 h), 48h (48 h) were cultured in PZM3 basal medium without IL17D, respectively, and then changed to PZM3 medium containing 50ng/mL IL17D, and the control NC group was continuously cultured with PZM3 culture without IL17D for all hours until 48h, 64h, and 168h to record cleavage number, 4 cell number, blastocyst number, and blastocyst cell number. Results as shown in table 2, cloned embryos cultured in PZM3 with IL17D added at 0h after activation had the best developmental efficiency, followed by 24h group, while IL17D added at 48h had no effect on embryo development.
TABLE 2 Effect of IL17D addition at various time periods after embryo activation on cloned embryo development efficiency
2.4 Effect of IL17D on blastocyst apoptosis. Cloned embryos treated with IL17D were collected for TUNEL and qPCR assays. The number level of blastula apoptotic cells in IL 17D-treated group was significantly reduced compared to the control group, and the results are shown in fig. 1 and 2. Further, qPCR (quantitative polymerase chain reaction) detects a plurality of apoptosis-related genes, and the results show that the expression of the apoptosis-promoting gene BCL2L11 of the blastula of the IL17D treatment group is down-regulated, while the expression of the apoptosis-inhibiting gene BCL2 is up-regulated, and the results are shown in FIG. 3. Therefore, IL17D can reduce apoptosis of embryonic cells, thereby improving development quality of cloned embryos and improving cloning efficiency.
What has been described above is but some implementations of the invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention. In addition, the above embodiments of the present invention can be used to improve the efficiency of the development and birth of cloned embryos of pigs, and also can be used to improve the efficiency of the development and birth of cloned embryos of other animals, such as mice, sheep, cattle, cats, etc., which also fall within the scope of the present invention.
Claims (10)
1. An additive for improving the development quality of a cloned embryo, wherein the additive comprises one or more cytokines of the IL17 family.
2. The additive for improving the development quality of cloned embryos of claim 1, wherein the additive is IL 17D.
3. The additive for improving the development quality of cloned embryos of claim 2, wherein the concentration of IL17D is 50-100 ng/mL.
4. The additive for improving the development quality of cloned embryos of claim 3, wherein the concentration of IL17D is 50 ng/mL.
5. The additive for improving the development quality of cloned embryos of claim 4, wherein the IL17D is added at 0h after the activation of cloned embryos.
6. Use of the additive of any one of claims 1-5 for improving the quality of cloned embryo development.
7. Use of the additive according to any one of claims 1 to 5 in the field of increasing the efficiency of animal cloning.
8. Use of the additive of any one of claims 1-5 for inhibiting apoptosis in cultured embryonic cells in vitro.
9. A method for increasing the efficiency of animal cloning using the additive of any one of claims 1-5, wherein the method comprises:
preparing fusion activated cloned and reconstructed embryos;
culturing the cloned reconstructed embryos 0h after activation in PZM3 basal medium containing 50ng/mLIL 17D;
placing the cloned reconstructed embryo cultured and developed to 2 cell stage into the uterus of surrogate pregnant animal;
the cloned animal is bred, and the birth efficiency of the cloned animal is improved.
10. The method of claim 9, wherein the method is suitable for use with swine, cattle, sheep, mice or other mammals.
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