High-efficiency C57BL / 6 mouse in-vitro fertilization method

Abstract

The invention discloses a high-efficiency C57BL / 6 mouse in-vitro fertilization method, relates to use of an in-vitro fertilization method in the technical field of biology, and particularly relates to a method for obtaining more high-quality fertilized eggs for gene editing in an in-vitro fertilization process of small animal gene editing model construction. Therefore, the animal cost of gene editing is greatly reduced, and wide application is promoted.

Description

Technical Field

[0001] This invention relates to the field of in vitro fertilization technology, specifically to a highly efficient method for in vitro fertilization of C57BL / 6 mice. Technical Background Currently, the construction of small animal gene editing models mainly involves obtaining fertilized eggs through in vitro fertilization (IVF) for editing. The success of IVF is affected by various factors, such as prolonged egg retrieval time leading to reduced egg motility; poor control of sperm capacitation time resulting in reduced motility; and cumbersome procedures reducing fertilization rates. The purpose of this invention is to rapidly obtain sperm and eggs, simplify the procedure, and perform IVF to obtain a high number of highly viable fertilized eggs. Summary of the Invention

[0002] The purpose of this invention is to provide a highly efficient method for in vitro fertilization of C57BL / 6 mice to solve the problems existing in the prior art.

[0003] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A highly efficient method for in vitro fertilization of C57BL / 6 mice, characterized by comprising the following steps: (1) Sperm preparation: male rat sperm was added to TYH culture medium and placed in a CO2 incubator for energy acquisition and use. (2) Egg preparation: Take the egg masses of the female mouse and drag them into HTF droplets for later use; (3) Fertilization: After capacitation, sperm are added to a fertilization dish containing cumulus ova, gently shaken, and then the fertilization dish is placed in an incubator for culture; (4) Culture: Observe whether the two pronuclei are present in the fertilized egg and whether the second polar body is released; wash the egg with HTF to remove excess sperm and fragments, and then put it in an incubator for overnight culture.

[0004] As an optimization, the following steps are included: (1) Sperm preparation: Make a 45μl drop of TYH in a 35mm culture dish, add 4.5ml of paraffin oil for oil sealing, then add another 45μl of TYH to the TYH drop, and preheat in a three-gas incubator for 20min to obtain a sperm capacitation dish; select male mice with normal reproductive capacity that are 10 weeks old or older, anesthetize them with CO2, remove the epididymal tails and vas deferens on both sides, remove the surrounding adipose tissue, pinch the epididymal tail tightly with the thumb and forefinger, gently puncture the epididymal tail with a sterile 1mL syringe needle, gently squeeze the epididymal tail to make the sperm flow out and add it to the TYH in the sperm capacitation dish, and place it in a CO2 incubator for capacitation for 30min; (2) Oocyte preparation: Two separate 180μl HTF droplets were prepared in a 35mm culture dish, 4ml of paraffin oil was added for oil sealing, and then the dish was placed in a three-gas incubator for preheating for at least 20min to obtain a fertilization dish; healthy female mice of 3-4 weeks were selected for superovulation, and 7U of PMSG hormone was injected intraperitoneally three days before in vitro fertilization, and 7U of HCG hormone was injected intraperitoneally 48-51h later; 13.5h after the female mice were injected with ovulation-inducing hormone HCG, they were anesthetized with CO2 and killed, and the ovaries and fallopian tubes were quickly dissected. The ampulla was torn in the paraffin oil in the fertilization dish to allow the cumulus to flow out, and then dragged into the HTF droplets in the fertilization dish. All the cumulus of 5 female mice were added to each HTF droplet; (3) Fertilization: Aspirate the sperm from the upper layer of the droplet in the sperm capacitation dish and add it to the droplet in the fertilization dish containing the cumulus ovum. Gently shake the dish and then place it in a 37°C, 5% CO2, 95% O2 incubator for 4-6 hours. (4) Culture: Observe whether the two pronuclei are present in the fertilized egg and whether the second polar body is released; wash the egg with HTF to remove excess sperm and fragments, and then put it in an incubator for overnight culture.

[0005] Compared with the prior art, the beneficial effects achieved by the present invention are: This invention improves upon existing technology and optimizes the fertilization process. The sperm collection process eliminates the need for fresh sperm dishes, reducing reagent consumption and saving time. Sperm squeezed directly from the epididymal tail is collected using tweezers, avoiding the problem of tissue fragments contaminating sperm caused by cutting the epididymal tail and vas deferens in the fresh sperm dish, as is done in existing technologies. This improves the success rate of in vitro fertilization in mice. Attached Figure Description

[0006] Figure 1 This is a flowchart of Embodiment 1 of the present invention; Figure 2 This is a cleavage diagram of Embodiment 1 of the present invention; Figure 3 This is an enlarged cleavage diagram of an egg from Embodiment 1 of the present invention; Figure 4 This is a cleavage diagram of the prior art of this invention. Detailed Implementation

[0007] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0008] To more clearly illustrate the method provided by the present invention, the following embodiments will be described in detail.

[0009] The sources of the raw materials and equipment used in the examples are as follows:

[0010] Example 1 A highly efficient in vitro fertilization method for C57BL / 6 mice mainly includes the following preparation steps: (1) Sperm preparation: Make a 45μl drop of TYH in a 35mm culture dish, add 4.5ml of paraffin oil for oil sealing, then add another 45μl of TYH to the TYH drop, and preheat in a three-gas incubator for 20min to obtain a sperm capacitation dish; select male mice with normal reproductive capacity that are 10 weeks old or older, anesthetize them with CO2, remove the epididymal tails and vas deferens on both sides, remove the surrounding adipose tissue, pinch the epididymal tail tightly with the thumb and forefinger, gently puncture the epididymal tail with a sterile 1mL syringe needle, gently squeeze the epididymal tail to make the sperm flow out and add it to the TYH in the sperm capacitation dish, and place it in a CO2 incubator for capacitation for 30min; (2) Oocyte preparation: Two separate 180μl HTF droplets were prepared in a 35mm culture dish, 4ml of paraffin oil was added for oil sealing, and then the dish was placed in a three-gas incubator for preheating for at least 20min to obtain a fertilization dish; healthy female mice at 4 weeks of age were selected for superovulation, and 7U of PMSG hormone was injected intraperitoneally three days before in vitro fertilization, and 7U of HCG hormone was injected intraperitoneally 50h later; 13.5h after the female mice were injected with ovulation-inducing hormone HCG, they were anesthetized with CO2 and killed, and the ovaries and fallopian tubes were quickly dissected. The ampulla was torn in the paraffin oil in the fertilization dish to allow the cumulus to flow out, and then dragged into the HTF droplets in the fertilization dish. All the cumulus of 5 female mice were added to each HTF droplet; (3) Fertilization: Aspirate the sperm from the upper layer of the droplet in the sperm capacitation dish and the droplet in the fertilization dish containing the cumulus of eggs, gently shake it, and then place the fertilization dish in a 37°C, 5% CO2, 95% O2 incubator for 6 hours; (4) Culture: Observe whether the two pronuclei are present in the fertilized egg and whether the second polar body is released; wash the egg with HTF to remove excess sperm and fragments, and then put it in an incubator for overnight culture.

[0011] Test Example 1 Five experiments were conducted according to the steps of Example 1. Fertilization was observed in step (4), and embryo cleavage was observed on the morning of the second day. The results are shown in Table 1.

[0012] Table 1

[0013] Two experiments were conducted following the methods described in the references. The epididymal tail and vas deferens were isolated, and fat and blood vessels were removed as much as possible. They were placed in a fresh semen dish, and the epididymal tail was cut 5-7 times with a syringe needle attached to a 30-gauge needle. Sperm was then gently squeezed out of the vas deferens with forceps (refer to Chapter 14, Section 14.3.2 of "Mouse Embryo Experimental Manual (Third Edition)"). The results of fertilization and embryo cleavage observations are shown in Table 2.

[0014] Table 2

[0015] As can be seen from the comparison results in Tables 1 and 2, Example 1 has a higher cleavage rate.

[0016] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of high efficiency in vitro fertilization of C57BL / 6 mice, characterized in that, The method comprises the following steps: (1) Sperm preparation: take the sperm of male mice and add to TYH culture solution, and put into CO2 incubator for capacitation; (2) Oocyte preparation: take the cumulus mass of female mice and drag into HTF droplet for standby; (3) Fertilization: take the capacitated sperm and add to the fertilization dish containing the cumulus mass, shake gently, and then put the fertilization dish into the incubator for culture; (4) Culture: observe whether two pronuclei exist in the fertilized oocyte and whether the second polar body is discharged; wash the oocyte with HTF to remove the excess sperm and debris, and then put into the incubator for overnight culture.

2. The method of high efficiency in vitro fertilization of C57BL / 6 mice according to claim 1, characterized in that, The method comprises the following steps: (1) Sperm preparation: select the male mice with normal breeding ability for more than 10 weeks, and kill by CO2 anesthesia, take out the cauda epididymidis and vas deferens, remove the surrounding adipose tissue, etc., pinch the cauda epididymidis tightly with the thumb and index finger, gently prick the cauda epididymidis with a sterile 1 mL syringe needle, and gently squeeze the cauda epididymidis to make the sperm flow out and add to the TYH in the sperm capacitation dish, and put into the CO2 incubator for capacitation for 30 min; (2) Oocyte preparation: select the healthy female mice for 3-4 weeks for superovulation, inject 7U of PMSG hormone into the abdominal cavity three days before in vitro fertilization, and inject 7U of HCG hormone into the abdominal cavity after 48-51 h; after the female mice are injected with the ovulation-promoting hormone HCG for 13.5 h, kill by CO2 anesthesia, quickly peel off the ovary and oviduct, tear the ampulla part in the paraffin oil in the fertilization dish to make the cumulus mass flow out, and then drag into the HTF droplet in the fertilization dish, and add 5 female mice's all cumulus masses to each HTF droplet; (3) Fertilization: take the sperm on the upper layer of the liquid droplet in the sperm capacitation dish and add to the liquid droplet containing the cumulus mass in the fertilization dish, shake gently, and then put the fertilization dish into the 37°C, 5% CO2, 95% O2 incubator for culture for 4-6 h; (4) Culture: observe whether two pronuclei exist in the fertilized oocyte and whether the second polar body is discharged; wash the oocyte with HTF to remove the excess sperm and debris, and then put into the incubator for overnight culture.

3. The method of high efficiency in vitro fertilization of C57BL / 6 mice according to claim 1, characterized in that, The preparation method of the sperm capacitation dish in step (1) is as follows: make a 45 μl TYH droplet in a 35 mm culture dish, add 4.5 ml paraffin oil for oil sealing, then add 45 μl TYH in the TYH droplet, and put into the three-gas incubator for preheating for 20 min.

4. The method of high efficiency in vitro fertilization of C57BL / 6 mice according to claim 1, characterized in that, The preparation method of the fertilization dish in step (2) is as follows: make two separate 180 μl HTF droplets in a 35 mm culture dish, add 4 ml paraffin oil for oil sealing, and then put into the three-gas incubator for preheating for at least 20 min.

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