A method for improving the birth rate of cloned mice by reconstructing nuclear transplanted blastocysts

Abstract

The present invention discloses a method for improving the birth rate of cloned mice by reconstructing nuclear transplanted blastocysts, which mainly collects 2-cell stage embryos from nuclear transplantation sources and performs 2i treatment to obtain the induced primitive ectoderm cell lineage from nuclear transplantation sources, and performs F4H treatment on 2-cell stage embryos from fertilization sources to obtain the induced primitive endoderm cell lineage from fertilization sources, and at the same time, the 2-cell stage embryos collected from the fertilization sources are electrically fused to prepare tetraploid embryos, and then the three cell lineages are reconstructed in vitro to obtain nuclear transplanted blastocyst cell lineage reconstructed blastocysts. After the nuclear transplanted cell lineage reconstructed blastocysts are transplanted into the uterus of surrogate mother mice, the birth efficiency of cloned mice is significantly improved. The reconstruction of the three cell lineages of nuclear transplanted blastocysts can not only improve the birth rate of cloned animals, but also provide a research scheme for studying how the interaction between the three cell lineages of post-implantation embryos affects embryo implantation and subsequent development.

Description

Technical Field

[0001] The present invention relates to the field of embryonic development, and in particular to a technical method for improving mouse cloning efficiency by reconstructing three cell lineages of mouse nuclear transplant blastocysts. Background Art

[0002] Mammalian somatic cell nuclear transfer is a technology that reprograms differentiated somatic cells into omnipotent embryos. Its application in the protection of endangered animals, therapeutic cloning, and the development of animal husbandry shows great potential. Although different species have been successfully cloned through this technology, there is always a serious problem: the efficiency of cloning is low. It is generally believed that it is caused by incomplete somatic cell reprogramming or epigenetic abnormalities in embryos during the preimplantation period, such as abnormal DNA methylation, acetylation, and loss of imprinted genes. During the development of nuclear transplanted embryos, various abnormalities occur at different stages. Compared with fertilized eggs, the initial translation of proteins in the cloned 1-2-cell embryonic period is insufficient, RNA processing and modification are lacking in the 2-4-cell embryonic period, abnormal transcription of some genes in the blastocyst period, abnormal establishment of H3K9me3 in the nuclear transplanted blastocyst during lineage specification, and abnormal WNT signaling pathways seriously hinder the development of cloned embryos and cloning efficiency. The cell lineage reprogramming of cloned embryos also profoundly affects their developmental outcomes, among which defects in the trophectoderm are an important reason for the low birth rate of cloned animals.

[0003] Regarding how to improve the efficiency of mouse cloning technology, most studies focus on improving epigenetic abnormalities. Adding deacetylase inhibitors such as TSA during the activation of nuclear transplanted embryos can reduce abnormal acetylation levels and increase the development rate of cloned embryos; silencing or knocking out XIST in donor cells can significantly increase the birth rate of cloned mice to 10%; injecting KDM4D and KDM5D during the 1-cell embryo stage can also significantly increase the blastocyst rate of cloned embryos. If combined with XIST knockout, the birth rate of cloned mice will increase to 23%. Reducing the level of abnormal DNA remethylation during the 4-cell embryo stage can also improve cloning efficiency. In addition to intervening in epigenetic abnormalities during the development of cloned embryos, embryo aggregation has been reported to improve cloning efficiency. The aggregation of clone-clone embryos increases the number of cells in cloned blastocysts, thereby improving the birth rate of cloned animals. The aggregation of clone-tetraploid embryos (fertilization source) also improves the development efficiency of cloned embryos, increasing the birth rate of cloned mice from 2.7% to 7.1%. According to existing studies, tetraploid embryos (4N BDF2) have a developmental fate similar to that of trophectoderm cells in late blastocysts, thus forming extraembryonic tissues that support embryonic development. The aggregation of tetraploid embryos and cell clusters in cloned blastocysts has increased the birth rate of cloned mice to 15%, and no large placenta syndrome has occurred.

[0004] Although the blastocyst rate of cloned embryos in vitro can be greatly improved according to the existing different strategies, there are still great differences in their developmental fate and pregnancy outcomes after implantation compared with fertilized eggs. This shows that the extremely high cloned blastocyst rate in vitro is not enough to support and save the developmental fate of cloned embryos after implantation, and in addition to the known epigenetic abnormalities, there are still unknown important factors that can regulate the development process of cloned embryos. It also highlights that the "black box" of cloned embryo development after implantation can no longer be explained and the mechanism explored only at the epigenetic level. At present, at the cell lineage level, defects in the trophectoderm and abnormal activation of the WNT signaling pathway in the primitive ectoderm in the three cell lineages of nuclear transplanted blastocysts limit the birth rate of cloned embryos. However, there are few studies on how the three cell lineages support the development of cloned embryos, and the relevant mechanisms are not clear. Therefore, it is urgent to explore how the three cell lineages coordinate with each other to ensure the development of cloned embryos and explore the corresponding intervention system. Summary of the invention

[0005] In order to solve or partially solve the problems existing in the related art, the present application provides a method for improving the efficiency of mouse cloning technology by reconstructing the lineage of blastocyst cells through nuclear transplantation.

[0006] The above method comprises the following steps:

[0007] (1) Mouse nuclear transplanted 2-cell embryos were cultured to the E4.5 blastocyst stage, and the trophectoderm cells were eliminated by removing the zona pellucida and immunosurgery, respectively, to obtain the inner cell mass, which is the induced primitive ectoderm derived from somatic cell nuclear transplantation;

[0008] (2) collecting 2-cell embryos from fertilization, culturing them to the E4.5 blastocyst stage, and eliminating the trophectoderm cells by removing the zona pellucida and immunosurgery to obtain the inner cell mass, which is the induced primitive endoderm from fertilization;

[0009] (3) taking 2-cell embryos from fertilization, preparing them into tetraploid embryos by electrofusion, and culturing them to 8-cell or mulberry stage embryos;

[0010] (4) The induced primitive ectoderm of (1), the induced primitive endoderm of (2), and the 8-cell or morula stage embryo of (3) are polymerized in a ratio of 1:1:2 to form a reconstructed blastocyst.

[0011] Furthermore, the mouse nuclear transfer 2-cell stage embryos in (1) were cultured in KSOM medium supplemented with 2i to obtain induced primitive ectoderm.

[0012] Furthermore, the 2-cell stage embryos derived from the fertilization in (2) were cultured in a KSOM medium supplemented with F4H to obtain induced primitive endoderm.

[0013] Further, the fertilized 2-cell stage embryos are electrofused to obtain tetraploid embryos, and cultured in the KSOM system to the 8-cell or mulberry stage to obtain 8-cell or mulberry stage embryos.

[0014] Furthermore, 2i is a small molecule compound that inhibits FGF / ERK signaling pathway and GSK3 signaling pathway, and 2i is PD0325901 and CHIR99021.

[0015] Further, F4H is FGF4 and heparin.

[0016] Furthermore, the polymerization in (4) was carried out at 37°C and 5% CO 2 carried out under conditions.

[0017] Beneficial Effects

[0018] The present invention reconstructs the cell lineage of mouse nuclear transplant blastocysts, compares the embryonic development potential before and after the reconstruction of the blastocyst lineage, and finds that in addition to the defect of the trophectoderm, the primitive endoderm cells of the mouse nuclear transplant blastocysts also have reprogramming defects. The reprogramming of the induced primitive endoderm and tetraploid embryos from fertilization is normal, so we use tetraploid embryos from fertilization to make up for the defect of the trophectoderm of the nuclear transplant blastocysts, and use the induced primitive endoderm from fertilization to replace the defective primitive endoderm cells of the nuclear transplant blastocysts, so that in the reconstructed nuclear transplant blastocysts, the reprogramming of the two cell lineages that develop into extraembryonic tissues is normal and sufficient to support the normal development of the embryo, thereby increasing the birth rate of cloned mice to 20.5%.

[0019] The present invention provides a method which can not only significantly improve the birth rate of cloned mice, but also has great potential in the protection of endangered animals, therapeutic cloning, animal husbandry development, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail the exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0021] Figure 1 :Reconstruction of three cell lineages in nuclear transplanted blastocysts;

[0022] A: Schematic diagram of cell lineage reconstruction;

[0023] B: 2i and F4H induce 2-cell development into E4.5 blastocysts, as well as blastocysts with successful lineage reconstruction.

[0024] Figure 2 : Flowchart of mouse somatic cell nuclear transfer.

[0025] Figure 3 :2-cell embryonic developmental fate under 2i and F4H induction;

[0026] A: Induction pattern diagram;

[0027] B: Immunofluorescence identification of the fate characteristics of 2-cell stage embryos under different induction methods, Nanog marks the primitive ectoderm cell lineage, and Gata6 marks the induced primitive endoderm cell lineage.

[0028] Figure 4 :Generation-reconstructed nuclear transfer blastocyst-derived cloned mice;

[0029] A: Birth rate, fetal and placental weights of reconstructed embryos;

[0030] B: Fetus born from reconstructed embryo, GFP indicates cells differentiated from iEPI, and td-tomato indicates cells derived from iPrE. DETAILED DESCRIPTION

[0031] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0032] Example

[0033] 1. Mouse nuclear transfer and acquisition of 2 cells from fertilization

[0034] 1. Mouse nuclear transplantation of 2 cells and acquisition of induced primitive outer layer cell lineages

[0035] Select 8-12 week old female mice of CAG-GFP-BDF1 and intraperitoneally inject 7.5 IU of pregnant mare serum gonadotropin (PMSG). 46-48 hours later, intraperitoneally inject 7.5 IU of human chorionic gonadotropin (HCG). 14 hours later, collect MII stage eggs and place them in the CZB prepared in advance. Incubate at 37°C, 5% CO 2 At the same time, the cumulus cells were collected into HCZB and temporarily stored at 4°C.

[0036] After the MII stage oocytes have recovered in CZB for 30 minutes, enucleation begins in the HCZB operating solution containing CB. There are 15-20 eggs in each group, and enucleation is completed in 5-10 minutes. The enucleated MII eggs are blown and transferred to a new CZB drop for recovery. After enucleation is completed, the cumulus cells that were previously collected and temporarily placed at 4°C are broken by a small pulse, and the cumulus cell nuclei are injected into the enucleated oocytes. After the injection is completed, the reconstructed 1-cell embryos are placed in CZB to recover for 1-2 hours, and then activated with strontium chloride for 6 hours, and then transferred to TSA (histone deacetylase inhibitor) for treatment for 3-4 hours, and finally placed in KSOM for continued culture to obtain nuclear transplanted 2-cell stage embryos ( Figure 2 ).

[0037] When the nuclear transplanted embryos developed to the 2-cell stage in vitro, the 2 cells with intact morphology were picked and continued to be cultured in the 2i system (3μM CHIR99021 (Axon #1386), 1μM PD0325901 (Stemgent 04-0006)) to the E4.5 stage. After culturing in the 2i-KSOM culture medium for 3 days, the nuclear transplanted 2 cells developed to the late blastocyst stage, and their inner cell mass changed to a state where only primitive ectoderm cells existed. After removing the trophectoderm cells of the late blastocyst with the help of immunosurgery, the inner cell mass obtained was the induced primitive ectoderm cells ( Figure 1 A) has a developmental fate of primitive ectoderm.

[0038] 2. Obtaining 2-cell embryos from fertilization and inducing primitive endoderm cell lineages

[0039] C57BL / 6 female mice of about 6 weeks old and expressing td-Tomato throughout the body were selected for superovulation, and then they were caged together with B6D2F1 male mice. The day when the plug was found was recorded as E0.5. The 2-cell stage embryos were collected at E1.5, and after recovering in KSOM culture medium for 1-2 hours, the 2-cell stage embryos were transferred to F4H-KSOM culture drops and continued to be cultured to the E4.5 stage. When the 2-cell stage embryos treated with 1000ng / ml FGF4 and 1μg / ml heparin (Heparin) developed to the late blastocyst stage, the inner cell mass cells in the blastocyst were composed only of primitive endoderm cells. The trophectoderm cells of the outermost layer of the blastocyst were removed by immunosurgery, and the separated inner cell mass was the induced primitive endoderm cells ( Figure 1 A) has a developmental fate similar to that of primitive endoderm cells.

[0040] 3. Obtaining Tetraploid Embryos

[0041] Take 8-12 weeks of female B6D2F1, after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) for superovulation, and then put them in the cage with B6D2F1 male mice. The next day, check the thrombus and record it as E0.5. At E1.5, collect 2-cell embryos of B6D2F2. After washing several times in KSOM culture medium, transfer the embryos to KSOM culture drops for recovery. Rinse the electrofusion tank with mouse embryo electrofusion solution, and add 30-40μl of electrofusion solution to the tank. After the instrument is connected, the 2-cell embryos are sucked out of the KSOM culture medium, electrofused and rinsed 3-4 times, and then placed in the electrofusion tank. When the embryo settles to the bottom, it is treated according to the set program. After the fusion program is completed, the embryos are taken out and placed in the incubator. The fused embryos are tetraploid embryos. After washing away the electrofusion solution in KSOM culture medium, the cells were transferred to new KSOM culture medium for continued culture, while the unfused embryos were discarded.

[0042] The procedure used for the electrofusion process of 2-cell embryos is: alternating current (AC) and direct current (DC). AC V: 20.0V, AC Time: 30s; DC V: 100V, OnTime: 40μs, OffTime: 0.50s, DC cycle: 1

[0043] 2. Immunofluorescence of induced primitive endoderm and induced primitive ectoderm

[0044] The 2-cell embryos from nuclear transfer were treated with 2i to obtain the induced primitive ectoderm cell lineage, and the fertilized 2-cell embryos were cultured under F4H conditions to obtain the induced primitive endoderm cell lineage. First, 2i and F4H were used to induce the 2-cells from fertilization, and the cell lineages generated by different induction methods were verified ( Figure 3A). Then, based on the known specific marker genes of the primitive endoderm and primitive ectoderm cell lineages in the blastocyst, the two cell lineages produced by the induction were immunostained to identify their molecular characteristics. The E4.5 blastocysts obtained by the control group (Control), 2i and F4H treatments were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.5% TritonX-100-PBS (PBST) at room temperature for 10 minutes, blocked with 2% BSA at room temperature for 0.5h, and incubated with 2% BSA at a dilution of 1:200 for the primary antibody, and incubated at 4°C overnight. The next day, the cells were washed three times with 0.5% PBST, and the secondary antibody was diluted at 1:500 with 0.5% PBST, and incubated at room temperature for 1.5-2h. After that, the cells were washed three times with PBST, DAPI was diluted at 1:3000 with PBST, treated in the dark for 5 minutes, and finally washed three times with 0.5% PBST. The immunostaining results of the three groups of embryos were observed under a confocal microscope. It was found that the inner cell mass of the blastocyst of the control group (Control) had two cell lineages, primitive ectoderm cells expressing the pluripotency gene Nanog and primitive endoderm cells expressing Gata6; the inner cell mass of the blastocyst obtained by 2i treatment only expressed Nanog, with characteristics similar to EPI; the inner cell mass of the blastocyst obtained by F4H treatment only expressed Gata6, with characteristics similar to PrE ( Figure 3 B). This indicates that 2i inhibits the formation of primitive endoderm in blastocysts, thereby acquiring induced primitive ectoderm cell lineage (iEPI), while F4H inhibits the formation of primitive ectoderm cell lineage in blastocysts, thereby acquiring induced primitive endoderm cell lineage (iPrE).

[0045] 3. Reconstruction of three cell lineages in nuclear transplanted blastocysts

[0046] 1. Cell lineage reconstruction

[0047] After obtaining the nuclear transplanted blastocysts treated with 2i (2i-NT-BL), the blastocysts derived from fertilization after F4H treatment (F4H-FD-BL), and the tetraploid embryos (FD-4n-8C), prepare for the reconstruction of the cell lineage. Use a polymerizing needle to make appropriate holes in the KSOM culture droplets in the culture dish, ensuring that the polymerizing holes are large enough to accommodate the cell clusters of the three cell lineages, and then place them in the incubator for preheating. Use hyaluronidase to remove the zona pellucida of the blastocyst and 8C embryo, and use immunosurgery to eliminate the trophectoderm cells of the blastocyst to expose the inner cell mass, and then use glass needles of different pore sizes to repeatedly suck and blow to completely peel off the inner cell mass. Figure 1As shown in A, the induced primitive ectoderm cell lineage (iEPI) and induced primitive endoderm cell lineage (iPrE) were obtained, and then aggregated with the 8-cell embryo of tetraploid B6D2F2 at a ratio of 1:1:2 to complete the reconstruction of the cell lineage. The embryos reconstructed by the blastocyst lineage were then placed in an incubator for further culture ( Figure 1 B).

[0048] 2. Dissection of E18.5 Fetuses for Lineage Reconstruction

[0049] The embryos obtained by nuclear transfer blastocyst cell lineage reconstruction (NT-iEPI+FD-iPrE+FD-4n) were cultured for 24 hours before embryo transplantation. As shown in Table 1, 18 E18.5 fetuses were obtained from the 88 transplanted nuclear transfer reconstructed blastocysts. As a control experiment, the cell lineages obtained from fertilized embryos induced by the 2i and F4H systems were reconstructed in the same way (FD-iEPI+FD-iPrE+FD-4n), and 55 reconstructed embryos were transplanted to obtain 12 E18.5 fetuses. Figure 4 As shown in B, NT-iEPI forms the fetus, FD-iPrE forms the fetal afterbirth, and the tetraploid embryo forms the placenta. After the nuclear transplanted embryos without lineage reconstruction were cultured in vitro to the late blastocyst stage, embryo transplantation was performed directly, and the birth rate of cloned mice was only 5%. The fetal birth rate and placental weight of E18.5 obtained from the three groups of nuclear transplantation, NT-iEPI+FD-iPrE+FD-4n and FD-iEPI+FD-iPrE+FD-4n were sorted and analyzed, and it was found that reconstructing the cell lineage of the nuclear transplanted blastocyst not only increased the birth rate of cloned mice, as high as 20.5%. In addition, the problem of large placenta that accompanies the birth of cloned mice was also solved ( Figure 4 A). Compared with the fetus obtained by direct transplantation of nuclear transfer blastocysts, the fetus obtained by nuclear transfer blastocyst cell lineage reconstruction (NT-iEPI+FD-iPrE+FD-4n) has a normal body weight.

[0050] Table 1 Different methods of embryo reconstruction and their in vivo development

[0051]

[0052] The above results indicate that we have successfully reconstructed the cell lineage of mouse nuclear transplant blastocysts. The nuclear transplant blastocysts with successfully reconstructed lineages subsequently have the developmental potential to give birth to individuals, and also improve the birth rate of cloned mice.

[0053] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for improving the birth rate of cloned mice by reconstructing nuclear transplanted blastocysts, characterized in that: The method comprises the following steps: (1) Mouse nuclear transplanted 2-cell embryos were cultured to the E4.5 blastocyst stage, and the zona pellucida and trophectoderm cells were removed to obtain the inner cell mass, which is the induced primitive ectoderm derived from somatic cell nuclear transplantation; The mouse nuclear transplantation 2-cell stage embryos are cultured in a system in which 2i is added to a KSOM culture medium to obtain an induced primitive ectoderm; the 2i is a small molecule compound that inhibits the FGF / ERK signaling pathway and the GSK3 signaling pathway, and the 2i is PD0325901 and CHIR99021; (2) Collecting 2-cell embryos from fertilization, culturing them to the E4.5 blastocyst stage, and removing the zona pellucida and trophectoderm cells to obtain the inner cell mass, which is the induced primitive endoderm from fertilization; The 2-cell stage embryos derived from the fertilization are cultured in a system in which F4H is added to KSOM culture medium to obtain induced primitive endoderm; the F4H is FGF4 and heparin; (3) Taking 2-cell embryos from fertilization, preparing them into tetraploid embryos by electrofusion, and culturing them to 8-cell or mulberry stage embryos; (4) The induced primitive ectoderm of (1), the induced primitive endoderm of (2) and the 8-cell or morula stage embryo of (3) are aggregated in a ratio of 1:1:2 to form a reconstructed blastocyst.

2. The method according to claim 1, characterized in that The 2-cell stage embryos derived from the fertilization are tetraploid embryos obtained by electric fusion, and are cultured in a KSOM system to the 8-cell or mulberry stage to obtain 8-cell or mulberry stage embryos.

3. The method according to claim 1, characterized in that The polymerization in (4) is carried out at 37°C and 5% CO2.

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