CN113403259B - An additive for improving the developmental quality of cloned embryos and its application - Google Patents

Abstract

The invention discloses an additive for improving the development quality of cloned embryos, which inhibits 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 further serves the fields of animal production, gene editing research and the like.

Description

An additive for improving the developmental quality of cloned embryos and its application

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 its application.

Background technique

The somatic cell nuclear transfer (SCNT) technology, also known as cloning technology, is widely used in livestock production, gene editing research, therapeutic cloning and other fields. In animal husbandry, cloning technology has been applied to the propagation of fine breeding stock, the production of bioreactors, and the breeding of food-saving, environmentally friendly and disease-resistant varieties. However, the low developmental efficiency of SCNT embryos has been the main reason limiting its expanded application. Among the many reasons that lead to the low development efficiency of SCNT embryos, the developmental arrest caused by early embryonic cell apoptosis has always been an important reason. Apoptosis of embryonic cells is beneficial for the removal of abnormally developed cells, but apoptosis beyond a certain level can lead to arrest of embryonic development. A large number of studies have shown that excessive apoptosis is observed in embryos before attachment, and apoptotic cells shrink, chromatin condensation, and DNA fragmentation. The causes of excessive apoptosis in SCNT embryos include abnormal zygote activation, abnormal segregation of chromosomes, oocytes, in vitro operating procedures, and culture environments. Studies have shown that adding the cytokine IGF1, the hormone melatonin, and vitamin C can inhibit the apoptosis of cloned embryonic cells and improve the developmental efficiency of cloned embryos.

Interleukin 17 (interleukin 17, IL17) is a family of cytokines. At present, six members including IL-17A-F have been identified and cloned through genome sequencing and proteomics. Existing studies have shown that IL17 family proteins mainly play a role in immunity, mainly by inducing cells to express pro-inflammatory cytokines, chemokines, and antimicrobial peptides, enhancing cellular immune responses, participating in inflammation and mucosal host defense, and playing a role in bacterial infection, It also has a certain function in tumorigenesis.

Contents of the invention

The object of the present invention is to provide an additive for improving the developmental quality of cloned embryos and its application in the field of improving the developmental quality of cloned embryos, improving the efficiency of animal cloning, and inhibiting the apoptosis of in vitro cultured embryonic cells.

One aspect of the present invention provides an additive for improving the developmental quality of cloned embryos, the additive includes one or more cytokines in the IL17 family.

In certain embodiments, the additive to improve the developmental quality of cloned embryos is IL17D.

In certain embodiments, the concentration of the additive IL17D for improving the developmental quality of cloned embryos is 50-100 ng/mL.

In some embodiments, the concentration of the additive IL17D for improving the developmental quality of cloned embryos is 50 ng/mL.

In some embodiments, the additive IL17D for improving the developmental quality of cloned embryos is added at 0 h after the cloned embryos are activated.

Another invention of the present invention provides an application of an additive for improving the development quality of cloned embryos in improving the development quality of cloned embryos. The additive is one or more cytokines in the IL17 family; preferably, the additive is IL17D; 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 added at 0h after activation of the cloned embryo.

The third aspect of the present invention provides an application of an additive for improving the developmental quality of cloned embryos in the field of improving animal cloning efficiency. The additive is one or more cytokines in the IL17 family; preferably, the additive is IL17D; 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 added at 0h after activation of the cloned embryo.

The fourth aspect of the present invention provides an application of an additive for improving the developmental quality of cloned embryos in inhibiting apoptosis of embryonic cells cultured in vitro. The additive is one or more cytokines in the IL17 family; preferably, the additive is IL17D; 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 added at 0h after activation of the cloned embryo.

The fifth aspect of the present invention provides a method for improving the efficiency of animal cloning by using additives that improve the developmental quality of cloned embryos, wherein the method includes: preparing fusion-activated cloned reconstituted embryos; The reconstructed embryos were cultured in PZM3 basal medium containing 50ng/mL IL17D; the cloned reconstructed embryos developed to the 2-cell stage were placed in the uterus of surrogate animals; cloned animals were bred to improve the birth rate and health of cloned animals. Aberdeen. The additive is one or more cytokines in the IL17 family; preferably, the additive is IL17D; 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 added at 0h after activation of the cloned embryo.

In some embodiments, the method of improving animal cloning efficiency by using additives that improve the developmental quality of cloned embryos is applicable to pigs, cows, sheep, mice or other livestock mammals.

Beneficial effects of the present invention: adding IL17D to the culture medium of in vitro cloned embryos inhibits the apoptosis of embryonic cells, thereby improving the developmental quality of cloned embryos, and is applied to the field of improving the developmental quality of cloned embryos, improving the efficiency of animal cloning techniques, and inhibiting the development of embryos in vitro. Cultivate embryonic cell apoptosis and other fields, and then serve for wider application in animal husbandry production, gene editing research and other fields.

Description of drawings

Figure 1 shows the results of Hoechst33342 staining and cell apoptosis detection (TdT-mediated dUTPNick-End Labeling, TUNEL) results of cloned embryos.

Fig. 2 is the result of apoptosis of cloned embryos treated with IL17D.

Fig. 3 is the result of the relative expression of some apoptotic genes in cloned embryos treated with IL17D.

detailed description

The present invention will be described in further detail below in conjunction with specific embodiments.

1. Construction of porcine cloned embryos

Oocyte maturation: Store porcine ovaries in 37°C preheated saline containing penicillin and streptomycin sulfate, use a 18-gauge needle and syringe, select follicles with a diameter of about 5 mm to extract follicular fluid, and let the follicular fluid Naturally settle for 10 minutes at 38.5°C, remove the supernatant and resuspend in DPBS, repeat the above steps twice, take the resuspension and place it in a 10cm culture plate, observe under a stereoscope and select a suitable cumulus with a mouth pipette Cell-oocyte complexes (Cumulus oocyte complexes, COCs), the selected COCs were washed 3 times with maturation medium, then transferred to in vitro maturation medium that had been equilibrated for 4 hours, and covered with embryo grade mineral oil. The components of the in vitro maturation medium are as follows: basal medium 199 (Gibco), 1g/L polyvinyl alcohol, 3.05mM glucose, 0.91mM sodium pyruvate, 0.57nM cysteine, 0.5μg/mL luteinizing hormone, 0.5μg /mL follicle-stimulating hormone, 10ng/mL epidermal growth factor, 100mL/L porcine follicular fluid, 75μg/ml penicillin (penicillin G), and 50μg/ml streptomycin. COCs were cultured in an incubator at 38.5°C and 5% CO ₂ for 42-44h. Place the cultured COCs in a 1.5 mL centrifuge tube containing 1 mg/mL hyaluronidase in DPBS, and gently blow with a pipette gun to remove the cumulus cells in the COCs. Finally, the oocytes with uniform cytoplasm, round cell shape, clear egg space and clear polar body were selected as the materials for the nuclear transfer experiment.

Donor cell preparation: Fetal fibroblasts were isolated from 35-day-old pig fetuses, and the separated cells were frozen in liquid nitrogen. The fetal fibroblasts were taken out of the liquid nitrogen tank and recovered 2-3 days before the nuclear transfer experiment. , cultured in 12% FBS medium, cultured at 38.5°C, 5% CO ₂ medium to 90% confluency. Digest with trypsin and resuspend in micromanipulation solution for later use.

Activation of clonally reconstituted embryo fusion: Oocytes are enucleated by blind aspiration, removing the first polar body and its surrounding tissues. Under the microscope, the donor cells with better morphology were selected and their nuclei were injected into the yolk circumference of the enucleated oocyte from the original incision to obtain clonal reconstructed embryos. The cloned and reconstituted embryos were cultured in porcine zygote medium-3 (PZM3) medium at 38.5°C and 5% CO ₂ for 4 hours, and then fusion activation was performed. The composition of PZM3 culture solution is as follows: NaCl 108.00mM, KCl 10.00mM, KH ₂ PO ₄ 0.35mM, MgSO4·7H ₂ O 0.40mM, NaHCO ₃ 25.07mM, sodium pyruvate (Na-Pyruvate) 0.20mM, calcium lactate 2.00mM, Taurine (Hypotaurine) 5.00mM, L-glutamine 1.00mM, essential amino acid 20mL/L, non-essential amino acid 10mL/L. . Set the parameters of the electrofusion instrument to 150v/mm, 100μs, 2DC, clean the electrofusion tank with fusion-activation solution, add 500 μL of fusion activation solution, transfer 10 cloned and reconstructed embryos to the electrofusion tank, and rotate the eggs with a glass fine needle. The acceptor-nuclei-donor axis is perpendicular to the electrodes for fusion activation of clonal reconstituted embryos.

2. In vitro culture of pig cloned embryos and analysis of cloning efficiency results

2.1 Treatment of in vitro medium PZM3

Add 0ng/mL, 50ng/mL, and 100ng/mL of IL17D protein to the PZM3 basal culture medium, and incubate for 44h for later use.

2.2 Effects of IL17D supplementation on the development of cloned embryos at 0h (0 hour) after fusion activation

0h after the cloned embryos were activated, they were placed in the culture plate containing the in vitro medium PZM3 supplemented with IL17D. When the cloned embryos were cultured to 48h, 64h and 168h, the number of cleavage, 4-cell number and blastocyst number were recorded. The blastocysts were collected, put into DPBS containing 10 μg/ml Hoechst33342 and stained for 10 minutes, and then the number of cells in the blastocysts was observed under a fluorescent microscope. The results are shown in Table 1. The developmental efficiency of cloned embryos cultured in in vitro medium PZM3 supplemented with 50ng/mL or 100ng/mL IL17D was higher than that of the control group (0ng/mL IL17D). The early development efficiency of the cloned embryos was the highest, compared with the control group, its 4-cell rate increased by 37.7%, and the blastocyst rate increased by 66.2%.

Table 1 Effects of different concentrations of IL17D on the developmental efficiency of cloned embryos

2.3 Effects of IL17D supplementation at different time periods after embryo activation on the development of cloned embryos.

According to the results in 2.2, the 50ng/mL IL17D treatment group was the optimal concentration group. Therefore, after embryo activation, they were cultured in PZM3 basal medium without IL17D for 0h (0 hour), 24h (24 hours), 48h (48 hours), and then replaced with PZM3 medium containing 50ng/mL IL17D, the control group In the NC group, the PZM3 culture medium without IL17D was used throughout the whole process, and the number of cleavage, 4-cell number, blastocyst number and blastocyst cell number were recorded at 48h, 64h and 168h. The results are shown in Table 2. After activation, PZM3 cultured with IL17D added at 0h had the best developmental efficiency of cloned embryos, followed by the 24h group, while adding IL17D at 48h had no effect on embryo development.

Table 2 Effect of adding IL17D at different time periods after embryo activation on the developmental efficiency of cloned embryos

2.4 The effect of IL17D on blastocyst cell apoptosis. Cloned embryos treated with IL17D were collected for TUNEL and qPCR detection. Compared with the control group, the number of blastocyst apoptotic cells in the IL17D treatment group was significantly reduced, and the results are shown in Figures 1 and 2. Further qPCR detection of multiple apoptosis-related genes showed that the expression of the pro-apoptotic gene BCL2L11 was down-regulated in blastocysts treated with IL17D, while the expression of the anti-apoptotic gene BCL2 was up-regulated. The results are shown in Figure 3. It can be seen that IL17D can reduce the apoptosis of embryonic cells, thereby improving the developmental quality of cloned embryos and improving the cloning efficiency.

What has been described above are only some implementations of the present invention. Those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all belong to the protection scope of the present invention. In addition, the embodiments of the present invention described above can not only be used to improve the development and birth efficiency of pig cloned embryos, but also can be used to improve the development and birth efficiency of cloned embryos of other animals, such as mice, sheep, cattle and cats. Also all belong to the protection scope of the present invention.

Claims (3)

1. An application of an additive in improving the early development efficiency of pig cloned embryos, wherein the additive is IL17D, and the IL17D concentration is 50ng/mL, and the additive is added at 0h after activation of in vitro cloned embryo culture. 2. An application of an additive in inhibiting apoptosis of porcine embryo cells cultured in vitro, wherein the additive is IL17D, the concentration of IL17D is 50 ng/mL, and the additive is added at 0 h after activation of in vitro cloned embryo culture. 3. Utilize a kind of additive to improve the method for the early stage development efficiency of pig cloned embryo, wherein, described method comprises: Preparation of fusion activated porcine clone reconstituted embryos; Place the reconstituted pig clone embryos 0h after activation in the PZM3 basal medium containing 50ng/mL IL17D; The pig cloned reconstructed embryos developed to the 2-cell stage were placed in the pig uterus; Cloned pigs are bred to improve the birth efficiency of cloned pigs.

Images