Method for producing somatic cell cloned pig

Abstract

The invention relates to the technical field of biology, and discloses a method for producing somatic cell cloned pigs, which comprises the following steps: collecting oocytes from ovaries of sows before a puberty period, carrying out maturation culture in vitro to obtain oocytes capable of being used for nuclear transplantation, separating and culturing somatic cells from healthy pig fetuses or adult pigs with high-quality genes, and separating and culturing somatic cells to obtain somatic cell cloned pigs. Taking the oocytes as donor cells, and removing cell nucleuses of the oocytes by using a micromanipulation technology to obtain enucleated oocytes; compared with the prior art, the method has the advantages that the production cost is reduced, and the production efficiency of cloned pigs is further improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for producing somatic cell cloned pigs. Background Art

[0002] Production somatic cell cloning technology refers to the use of animal somatic cells, through the method of nuclear transplantation, the somatic cell nucleus is injected into the enucleated recipient cell, and then through appropriate stimulation and treatment, the recipient cell develops into an individual with exactly the same genes as the original somatic cell. This technology provides new treatment methods in the medical field, such as preparing cells and tissues for disease treatment, and is also an important means to study gene function and cell biology. With the advancement of biotechnology, animal cloning technology has shown great potential in many fields, but the traditional somatic cell cloned pig production method has challenges such as high production cost, low production efficiency, and high technical difficulty. The high investment in experimental materials and equipment, complex operating procedures, and low pregnancy and survival rates limit its application in large-scale production. The existing methods for producing somatic cell cloned pigs have the problems of high production cost and low production efficiency. Summary of the invention

[0003] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for producing somatic cell cloned pigs.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: a method for producing somatic cell cloned pigs, comprising the following steps:

[0005] Step 1: Collect oocytes from the ovaries of pre-pubertal sows and culture them in vitro to obtain oocytes that can be used for nuclear transplantation;

[0006] Step 2: Isolate and culture somatic cells from healthy and genetically high-quality pig fetuses or adult pigs as donor cells;

[0007] Step 3: Use micromanipulation technology to remove the nucleus of the oocyte to obtain an enucleated oocyte;

[0008] Step 4: Inject or fuse donor cells into enucleated oocytes through microinjection or electrofusion technology to form reconstructed embryos;

[0009] Step 5: The reconstructed embryos are chemically or physically activated and then cultured under specific in vitro culture conditions until they develop to the early embryonic stage;

[0010] Step six: Transplant the reconstructed embryo that has developed to the early embryonic stage into the uterus of a surrogate sow that is in synchronized estrus and in good physiological condition. After the gestation process, a somatic cell cloned pig is produced.

[0011] As a further description of the above technical solution:

[0012] The in vitro maturation culture conditions of oocytes include: appropriate temperature, humidity, pH value and specific culture medium formula, as well as possible addition of hormones and growth factors.

[0013] As a further description of the above technical solution:

[0014] The in vitro culture conditions of donor cells include: appropriate temperature, humidity, gas environment, culture medium formulation and possible addition of growth factors to ensure the activity and proliferation ability of the cells.

[0015] As a further description of the above technical solution:

[0016] Micromanipulation techniques include, but are not limited to, microdissection, microinjection, or laser enucleation techniques to ensure complete removal of the nucleus without damaging other structures of the oocyte.

[0017] As a further description of the above technical solution:

[0018] The formation of reconstructed embryos can be achieved by directly injecting the nucleus or cytoplasm of donor cells into enucleated oocytes through microinjection technology, or by fusing donor cells with enucleated oocytes through electrofusion technology to form reconstructed embryos.

[0019] As a further description of the above technical solution:

[0020] The specific in vitro culture conditions include, but are not limited to, appropriate temperature, humidity, gas environment, culture medium formulation, and possible addition of growth factors to simulate the environmental conditions for embryonic development in vivo.

[0021] As a further description of the above technical solution:

[0022] The surrogate sows are synchronized with their estrus to ensure that their developmental stage matches that of the reconstructed embryos, thereby improving the pregnancy rate and production efficiency after transplantation.

[0023] As a further description of the above technical solution:

[0024] The somatic cell cloned pigs produced are subjected to genotyping and health status assessment to ensure the accuracy of their genetic information and physiological health status.

[0025] The present invention has the following beneficial effects:

[0026] 1. In the present invention, porcine fetal fibroblasts are selected as donor cells. Such cells are not only easy to obtain, but also can be cultured for a long time in vitro. Compared with other types of donor cells, such as embryonic stem cells, porcine fetal fibroblasts do not require complicated culture and screening processes, which significantly reduces production costs.

[0027] 2. In the present invention, oocytes are collected from the ovaries of pre-pubertal sows, which reduces the difficulty of obtaining them. At the same time, the quality of these oocytes is also higher, further improving the production efficiency of cloned pigs. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] An embodiment provided by the present invention is a method for producing somatic cell cloned pigs, comprising the following steps:

[0030] Step 1: Collect oocytes from the ovaries of pre-pubertal sows and culture them in vitro to obtain oocytes that can be used for nuclear transplantation;

[0031] Step 2: Isolate and culture somatic cells from healthy and genetically high-quality pig fetuses or adult pigs as donor cells;

[0032] Step 3: Use micromanipulation technology to remove the nucleus of the oocyte to obtain an enucleated oocyte;

[0033] Step 4: Inject or fuse donor cells into enucleated oocytes through microinjection or electrofusion technology to form reconstructed embryos;

[0034] Step 5: The reconstructed embryos are chemically or physically activated and then cultured under specific in vitro culture conditions until they develop to the early embryonic stage;

[0035] Step six: Transplant the reconstructed embryo that has developed to the early embryonic stage into the uterus of a surrogate sow that is in synchronized estrus and in good physiological condition. After the gestation process, a somatic cell cloned pig is produced.

[0036] The in vitro maturation culture conditions of oocytes include: appropriate temperature, humidity, pH value and specific culture medium formula, as well as possible addition of hormones and growth factors;

[0037] The in vitro culture conditions of donor cells include: appropriate temperature, humidity, gas environment, culture medium formula and possible addition of growth factors to ensure cell activity and proliferation ability;

[0038] Micromanipulation techniques include, but are not limited to, microdissection, microinjection, or laser punch enucleation techniques to ensure complete removal of the nucleus without damaging other structures of the oocyte;

[0039] Reconstructed embryos can be formed by directly injecting the nucleus or cytoplasm of donor cells into enucleated oocytes through microinjection technology, or by fusing donor cells with enucleated oocytes through electrofusion technology to form reconstructed embryos;

[0040] Specific in vitro culture conditions include, but are not limited to, appropriate temperature, humidity, gas environment, culture medium formulation, and possible addition of growth factors to simulate the environmental conditions for embryonic development in vivo;

[0041] Synchronize estrus of surrogate sows to ensure that the developmental stage matches that of the reconstructed embryos, thus improving pregnancy rates and production efficiency after transplantation;

[0042] The somatic cell cloned pigs produced are subjected to genotyping and health status assessment to ensure the accuracy of their genetic information and physiological health status.

[0043] Working principle:

[0044] First, the acquisition and culture of oocytes

[0045] 1. Acquisition: Oocytes are taken from the ovaries of pre-pubertal sows to ensure the source and quality of the oocytes. Oocytes obtained from the ovaries of pre-pubertal sows are of higher quality and more suitable for subsequent nuclear transplantation operations.

[0046] 2. Culture: The retrieved oocytes are subjected to in vitro maturation culture to simulate the physiological environment in vivo, so that the oocytes can reach a mature state in vitro and provide high-quality recipient cells for subsequent nuclear transplantation.

[0047] Next, preparation of donor cells

[0048] 1. Selection: Choose healthy, high-quality porcine fetal fibroblasts as donor cells. Porcine fetal fibroblasts are easy to obtain and can be cultured in vitro for a long time to maintain cell activity and quantity.

[0049] 2. Culture: The selected donor cells are cultured in vitro to ensure the activity and quantity of the cells, providing a sufficient source of donor cells for subsequent nuclear transplantation.

[0050] Then, enucleate the oocyte

[0051] Using micromanipulation technology, the nucleus of the oocyte is removed. This step is one of the key steps in somatic cell cloning technology. It aims to clear the genetic material of the oocyte and prepare for subsequent nuclear transplantation.

[0052] Then nuclear transplantation

[0053] 1. Microinjection: Donor cells are injected into enucleated oocytes through microinjection to form reconstructed embryos. This step requires precise operating skills to ensure that the nucleus of the donor cell is successfully injected into the oocyte.

[0054] 2. Fusion: Observe under a microscope to confirm that the nucleus of the donor cell has successfully fused with the oocyte to form a reconstructed embryo.

[0055] Embryo activation and culture

[0056] 1. Activation treatment: The reconstructed embryos are activated to simulate the fertilization process in vivo, so that the reconstructed embryos begin to divide and develop.

[0057] 2. Cultivation under gas phase conditions: In vitro cultivation is carried out under gas phase conditions with an oxygen volume percentage of 5-10% to obtain early embryos. This gas phase condition has been optimized to increase the development rate of the embryos.

[0058] Finally, embryo transfer

[0059] The early embryos are transplanted into the fallopian tubes or uterus of pregnant sows. After the sows become pregnant, somatic cell cloned pigs can be produced.

[0060] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for producing somatic cell cloned pigs, characterized in that: The following steps are involved: Step 1: Collect oocytes from the ovaries of pre-pubertal sows and culture them in vitro to obtain oocytes that can be used for nuclear transplantation; Step 2: Isolate and culture somatic cells from healthy and genetically high-quality pig fetuses or adult pigs as donor cells; Step 3: Use micromanipulation technology to remove the nucleus of the oocyte to obtain an enucleated oocyte; Step 4: Inject or fuse donor cells into enucleated oocytes through microinjection or electrofusion technology to form reconstructed embryos; Step 5: The reconstructed embryos are chemically or physically activated and then cultured under specific in vitro culture conditions until they develop to the early embryonic stage; Step six: Transplant the reconstructed embryo that has developed to the early embryonic stage into the uterus of a surrogate sow that is in synchronized estrus and in good physiological condition. After the gestation process, a somatic cell cloned pig is produced.

2. A method for producing somatic cell cloned pigs according to claim 1, wherein in step 1, the in vitro maturation culture conditions of the oocytes include: Suitable temperature, humidity, pH value and specific culture medium formulation, as well as possible addition of hormones and growth factors.

3. A method for producing somatic cell cloned pigs according to claim 1 or 2, wherein in step 2, the in vitro culture conditions of the donor cells include: Appropriate temperature, humidity, gas environment, culture medium formulation and possible addition of growth factors to ensure cell activity and proliferation ability.

4. A method for producing somatic cell cloned pigs according to any of the preceding claims, wherein in step three, micromanipulation techniques include but are not limited to microdissection, microinjection or laser punching and enucleation techniques to ensure complete removal of the cell nucleus without damaging other structures of the oocyte.

5. A method for producing somatic cell cloned pigs according to any of the preceding claims, wherein in step 4, the formation of the reconstructed embryo can be achieved by directly injecting the nucleus or cytoplasm of the donor cell into the enucleated oocyte through microinjection technology, or by fusing the donor cell with the enucleated oocyte through electrofusion technology to form the reconstructed embryo.

6. A method for producing somatic cell cloned pigs according to any of the preceding claims, wherein in step 5, the specific in vitro culture conditions include but are not limited to suitable temperature, humidity, gas environment, culture medium formula and possible addition of growth factors to simulate the environmental conditions for embryonic development in vivo.

7. A method for producing somatic cell cloned pigs according to any of the preceding claims, further comprising performing estrus synchronization treatment on surrogate sows to ensure matching with the developmental stage of the reconstructed embryo and improve pregnancy rate and production efficiency after transplantation.

8. A method for producing somatic cell cloned pigs according to any of the preceding claims, further comprising genotyping and health status assessment of the produced somatic cell cloned pigs to ensure the accuracy of their genetic information and physiological health status.