KR20100053929A - Recombinant mouse interleukin-6 improve viability in mouse fertilized and cloned embryos - Google Patents

Abstract

PURPOSE: An in vitro culture system of mouse fertilized egg and cloned egg is provided to improve in vitro culture system and to enhance survival rate of the cells by adding a recombinant interleukin-6 to a basic medium(M16). CONSTITUTION: A mouse fertilized egg is cultured by adding recombinant mouse interleukin-6 to a basic medium(M16) and culturing initial fertilized egg and cloned egg in vitro without BSA(bovine serum albumin). The medium of the fertilized egg is M2 medium for washing and M16 medium for culturing.

Description

Improved incidence of mouse fertilized and cloned eggs by addition of recombinant mouse interleukin-6 in medium {Method of inhibiting apoptosis during in vitro culture of mouse nuclear transfer embryos {RECOMBINANT MOUSE INTERLEUKIN-6

The present invention relates to a method for in vitro culture of mouse fertilized eggs and cloned fertilized eggs, more specifically, by adding a recombinant mouse interleukin-6 to the basal medium, a more effective culture system compared with the existing in vitro culture system of mouse fertilized eggs and cloned fertilized eggs The present invention relates to a method for in vitro culture of mouse fertilized eggs and cloned fertilized eggs.

Recently, livestock transformation techniques and cloned animal production experiments have been conducted around the world. In these experiments, manipulating the fertilized egg in vitro is an essential process. Therefore, researches are being actively conducted to develop more effective and stable in vitro culture system.

At present, a method of incubating fertilized eggs in vitro and adding a protein additive such as serum to the basal medium is generally used. However, protein additives, such as serum, do not fully know their composition and do not know the cause of abnormal development by interaction with other compositions in the medium.

The present invention is to solve the problem of apoptosis and the resulting survival rate in in vitro culture of mouse fertilized eggs and cloned fertilized eggs.

In order to solve this problem, in the present invention, the fertilized egg and the cloned egg of the mouse are added to the existing basal medium (M16) by adding recombinant mouse Interleukin-6 (BioSource, Part No .: PMC0065, USA). By suppressing apoptosis, the survival rate of fertilized embryos up to blastocyst stage of early embryos was improved.

Therefore, by adding the recombinant mouse interleukin-6 to the basal medium, the in vitro culture method of the mouse fertilized egg and the cloned fertilized egg to provide a more effective culture system compared with the existing in vitro culture of mouse fertilized egg and cloned egg. It has the effect of enabling development and improving the survival rate of fertilized eggs.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by culturing the fertilized mouse to blastocyst by adding recombinant mouse Interleukin-6 (Part No .: PMC0065) to the basal medium.

In the conventional method, fertilized eggs have been cultured in a basal medium (M16) to which 0.4% bovine serum albumin or glutathione (GSH, Sigma, G2140) is added. However, the medium containing only bovine serum albumin (BSA, Sigma, A8806) does not inhibit apoptosis, and the development rate was poor when a chemical agent such as glutathione was used.

In the present invention, the fertilized egg of the mouse in the absence of bovine serum albumin (BSA, Sigma, A8806) in the basal medium (M16) recombinant mouse Interleukin-6 (Recombinant mouse Interleukin-6 US BioSource, Part No .: PMC0065) By the method of the present invention to add the not only the composition can be confirmed, but also the survival rate and cell death was significantly reduced.

The present invention provides an in vitro culture method for fertilized eggs which is not only easier to interpret the results than conventional fertilized egg culture methods using fetal bovine serum, but also has excellent efficiency.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.

Example 1 Production of Mouse Embryos

The medium for in vitro culture was prepared as follows.

Washing M2 medium (Sigma, Cat. No .: M7167);

Culture M16 medium (Sigma, Cat. No .: M7292);

The media were refrigerated.

Female mice were intraperitoneally injected with 5IU of pregnant mare's serum gonadotropin (PMSG), and mice injected with PMSG after 48 hours were intraperitoneally injected with 5IU of human chorionic gonadotropin (mCG) and then mated with male mice. It can be modified. After 12 hours, it was confirmed that the fertilization, these mice were cervical distal bone, and the dorsal incision was removed, washed and collected by collecting a fallopian tube in a petri dish prepared with a small amount of M2 medium. While looking under the microscope, the tip of the fallopian tube was grasped with forceps, the needle of the syringe was inserted into the hole, and slowly flushed with the M2 medium of the syringe to push out the fertilized egg in the fallopian tube. Fertilized eggs surrounded by mature cumulus cells were removed using hyaluronidase.

In vitro maturation medium, the temperature and the concentration of carbon dioxide were adjusted for 3 hours in an incubator at 37 ℃ with 5% of carbon dioxide before incubation of the egg. The collected eggs were placed in a mature medium in a 4-well dish and 50 eggs per well were added to control and recombinant mouse Interleukin-6 after 30 hours. -6 US BioSource, Part No .: PMC0065) was incubated for 4 days in a medium added.

Example 2 Production of Mouse Cloned Embryos

Step 1: Harvest mature eggs

Female mice were intraperitoneally injected with 5IU of pregnant mare's serum gonadotropin (PMSG) and 48 hours later, mice were injected with PMSG 12 hours after intraperitoneal injection of 5IU of human chorionic gonadotropin (hCG). These live rats were cervical vertebral, and the dorsal section was cut and the oviduct was removed and washed in a petri dish prepared with a small amount of M2 medium. While looking under the microscope, the tip of the fallopian tube was grasped with forceps, the needle of the syringe was inserted into the hole, and slowly flushed with the M2 medium of the syringe to push out the fertilized egg in the fallopian tube. Nucleated pulmonary eggs surrounded by mature cumulus cells were removed using hyaluronidase.

Put the lumps of eggs and oocytes in M2 medium (Sigma) containing 0.1% hyaluronidase, remove the oocytes by pipetting, wash only three eggs in M2 medium, and then carry out nuclear transfer with donor cells. Provided.

Second step: preparation of somatic cells

After collecting the oocytes removed in the first stage of oocyte harvesting, the cells were collected and centrifuged at 2000 rpm and washed three times with M2 medium to remove hyaluronates, and then the oocytes were provided by nuclear transplantation.

Third step: removing the oocytes and performing denucleation

For denuclearization, a microporous medium was prepared in a working dish with medium added with M2 medium (Sigma) and cytocalin B (cytochalasin B, CB, 5 ug / ml). The working dish was placed on the operation plate of the microinjector, and the fixing pipette was positioned at 9 o'clock, and the denuclearization pipette was placed at 3 o'clock. The fixed pipette was aspirated to fix the egg, and then the coarse and fine screw were used to focus the egg. The denuclear pipette was moved up and down so that the egg and the focal point were the same. The pipette for denuclearization was pierced through the zona pellucida at a suitable speed and force by using Piezo (Prime Tech, Japan), and removed by inhaling the cell nucleus and some cytoplasm around it. After denuclearization, the eggs were placed in an incubator and cultured for at least 20 minutes, and then used for transplantation of donor cells.

Step 4: transplanting donor cells prepared in advance into denuclearized eggs

For transplantation of donor cells, micronized with M2 medium containing 10% polyvinylpyrrokidone (FW. 360,000, Sigma) was placed on the operation panel of the microinjector and the donor cells prepared in step 2 Put into the enemy and mix. After the denucleated eggs were left in the incubator for 20 minutes or more, they were taken out and transferred to a micro medium of transplant medium, that is, M2 medium, using a washing pipette. After replacing the denuclear pipette with a transplant pipette, one or several donor cells were injected into the transplant pipette in M2 medium microfluidic medium containing 10% polyvinylpyrrolidone with donor cells. At this time, the donor cells were put into the pipette several times using a transplant pipette to spit out the cell membrane of the donor cell, and it was confirmed that the cell membrane is broken and debris is generated. The transplant pipette containing donor cells was then transferred to M2 medium with already denucleated eggs. The fixed pipette was aspirated in the same manner as in denuclearization, and the eggs were fixed a little, and then the coarse and fine screws were used to focus the eggs. The transplant pipette was moved up and down to bring the egg into focus. Implantable pipettes were made through a piezo (Prime Tech, Japan) to penetrate the zona pellucida at the proper speed and force, and then change the speed and force of the next piezo to the minimum. After transplanting the pipette, the medium inside the cell was removed, the fixation pipette was brought into close proximity, and the donor cells were injected per egg by breaking the cytoplasmic membrane using a piezo and applying hydraulic pressure to the cut site. After transplantation, the eggs were placed in M16 culture medium (M16, Sigma).

Step 5: Activation of the Reconstruction Column

The fused eggs were incubated for at least 30 minutes in in vitro culture medium until activated. Activation was incubated for 6 hours by adding 10 mM strontium chloride (SrCl _2, Sigma) and 5 μg / ㎖ CB to the calcium-free CZB medium (Table 2).

Sixth Step: Culture of Nuclear Transplanted Eggs

Reconstructed and activated embryos were washed three times in M16 medium and then cultured in a 37 ° C. incubator with 5% carbon dioxide in the same medium.

Example 3 Effect of Recombinant Mouse Interleukin-6 on the Development of Embryos

The fertilized eggs produced in Example 1 were cultured in the following medium.

(1) Basic medium (M16)

(2) Recombinant mouse Interleukin-6 (BioSource, USA, Part No .: PMC0065) of basal medium (M16) + 10ng / ml

(3) Recombinant mouse Interleukin-6 (BioSource of USA, Part No .: PMC0065) of basal medium (M16) + 100ng / ml

Two-cell-cytoplasmic mouse embryos were selected and further cultured in each of the above mediums for 4 days. After 4 days, the development rate up to blastocysts in each medium was examined.

Example 4 Effect of Recombinant Mouse Interleukin-6 on the Development of Mouse Cloned Embryos

Replication embryos produced in Example 2 were each cultured in the medium of the same treatment group as in Example 3.

The cloned eggs were incubated for 6 hours in an in vitro culture medium containing 5 μg / ml CB, and the nuclear transfer embryos were incubated for 4 days in a saturated humidity incubator of 37 ° C. and 5% carbon dioxide in the above three media, respectively. In order to investigate the effects of Recombinant mouse Interleukin-6 (BioSource, Part No .: PMC0065) on the development of cloned eggs, recombinant mouse interleukin-6 was added to the in vitro medium. On day 4, the development rate up to blastocysts in the treated and control groups was confirmed.

Example 5 Effect of Recombinant Mouse Interleukin-6 on Cell Number and Apoptosis of Blastocysts

After culturing for 5 days, the blastocysts were recovered and fixed in 3.7% formalin solution for 10 minutes, and then 2.5 mg / ml sodium azide and 2.5 μg / ml hoist (Hoechst 33342, Sigma, Cat. It was stained with a solution of glycerol (3): PBS (1) containing No.:B2261).

The egg dyeing process is as follows.

Paraffin droplets were formed in four places on one-third of the slide glass, and then 4 to 10 eggs were placed in the middle of the four spots and the cover glass was covered. Next, the egg was pressed using a thin object such as a ballpoint pen or a pipette tip. Then, the solution containing the hoist (Hoechst 33342 Sigma, Cat. No.:B2261) was flowed and the filter paper was passed on the opposite side to pass the solution.

In situ Cell Death Detection Kit was used to investigate the cell death rate of the blastocyst stage of porcine unit embryos cultured in the above three media. After 1 hour fixation in 3.7% formalin solution on day 5 of in vitro culture, the cells were washed with phosphate buffer solution (PBS) containing polyvinylpyrrolidone (PVP). And infiltrated at 0.5% Triton-X-100 for 1 hour, followed by washing and fluorescein-conjugated deoxyuridine triphosphate (dUTP) and terminal deoxynucleotidyltransferase enzyme (terminal). deoxynucleotidyl transferase enzyme) was reacted at 37 ° C. in the dark for 1 hour. In addition, 40 μg / ml propidium iodide (PI) and 40 μg / ml RNase A were treated to confirm cell viability and reacted at 37 ° C. in the dark for 1 hour, followed by fluorescence microscopy.

Example 6 Effect of Recombinant Mouse Interleukin-6 on Gene Expression Related to Apoptosis

The fertilized eggs developed up to blastocyst were collected from the control and treatment groups, respectively, and collected in tubes. It was then rapidly frozen in liquid nitrogen and stored at -70 ° C until analysis. Oocyte mRNA was isolated according to the company's protocol using the Dynabeads mRNA Direct Kit (Dynal Asa, Oslo, Norway). cDNA was synthesized from extracted mRNA using Oligo (dT) 12-18 (Invitrogen Co., Grand Island, NY) and Superscript reverse transcriptase enzyme (Invitrogen Co., Grand Island, NY). It was. In this experiment, the relative expression level of each gene was determined using histomic H2a mRNA (histome H2a mRNA) as an internal standard. Next, amplification was performed 40 times by real time reverse transcriptase polymerase chain reaction.

After the analysis was repeated three times, the significance was verified by the Turkey's Multiple Range Test of the SAS GLM program. Data are expressed as mean ± standard error, and P <0.05 is considered significant.

Table 1 below shows the composition of the basal culture (M16 medium) for in vitro culture of mouse somatic cell nuclear transfer embryos.

ingredient Concentration (g / ℓ) Calcium chloride (2H ₂ O) 0.25137 Magnesium sulfate anhydrous 0.1649 Potassium chloride 0.35635 Potassium Dihydrogen Phosphate (potassium phosphate monobasic) 0.162 Sodium bicarbonate 2.101 Sodium chloride 5.53193 Albumin (Bovine Fraction V) 4.0 D-glucose 1.0 Phenolic red sodium 0.0106 Sodium pyruvate (Na) 0.0363 DL-lactic acid (Na) 3.31985

Table 2 below shows the composition of the activating medium (Ca ²⁺ -removed CZB) of mouse somatic cell nuclear transfer embryos.

ingredient density Magnesium sulfate anhydrous 1.18mM Potassium chloride 4.83mM Potassium Dihydrogen Phosphate (potassium phosphate monobasic) 1.18mM Ethylenediaminetetraacetate (EDTA) 0.11 mM Sodium bicarbonate 25.12 mM Sodium chloride 81.62mM Albumin (Bovine Fraction V) 0.04% Sodium pyruvate (Na) 0.27 mM DL-lactic acid (Na) 31.30mM

As a result of incubating the fertilized eggs and cloned eggs produced in Examples 3 and 4 in the above three types of medium for 5 days, there was no difference in the development rate of mouse fertilized eggs as shown in Table 3 below (Table 3; basic medium). (M16) effect on the development rate of interleukin-6 fertilized egg), cloned fertilized egg was improved in the developmental group to the blastocyst in the treatment group added 100ng / ㎖ of recombinant mouse interleukin-6 (P <0.05, Table 4: Basic medium (effect on the development rate of cloned embryos of interleukin-6 in M16).

badge Egg number Blastocyst development rate (%) M16 382 98.31 ± 5.12 M16 + 10ng / ml Interleukin-6 382 97.22 ± 6.02 M16 + 100ng / ml Interleukin-6 371 98.83 ± 5.42

badge Egg number Blastocyst development rate (%) M16 98 45.1 ± 4.21 M16 + 10ng / ml Interleukin-6 89 57.1 ± 3.21 * M16 + 100ng / ml Interleukin-6 91 55.9 ± 2.17 *  * P <0.05

As a result of culturing the mouse fertilized egg produced in Example 5 in the above two medium, as shown in Table 5 (effect on the blastocyst cell number of interleukin-6 in basal medium (M16)), interleukin- for 5 days. The effect of increasing cell viability in medium supplemented with 6 showed a significantly higher survival rate of blastocysts, ie, a higher number of cells in blastocysts, when 100 ng / ml of interleukin-6 was treated in the medium (Table 5). In addition, apoptosis was shown to be markedly reduced (Table 6; effect on the suppression of apoptosis of blastocyst embryos of interleukin-6 in basal medium (M16)).

As a result of culturing the mouse fertilized egg produced in Example 6 in the above three media, the apoptosis gene of interleukin-6 as shown in Table 7 (influence on gene expression related to apoptosis of blastocysts by interleukin-6) The effect on the expression showed that the expression of genes that induce apoptosis was significantly lower (P <0.05) when 100ng / ml interleukin-6 was treated in the basal medium.

badge Repeat count Blastocyst development rate (%) M16 5 1.00 ± 0.00 M16 + 10ng / ml Interleukin-6 5 0.87 ± 0.07 M16 + 100ng / ml Interleukin-6 5 0.36 ± 0.04 *  * P <0.05

As described above, the present invention provides cell viability (cells in blastocysts) by adding recombinant mouse Interleukin-6 (Part No .: PMC0065) when culturing mouse fertilized eggs and cloned eggs in vitro. It provides a simple culture method that can increase the number of cells), inhibit apoptosis, and reduce the expression of genes that induce apoptosis and develop into higher quality blastocysts. In addition, the present invention shows a clearer result than the culturing method by the addition of protein mainly used until now. Therefore, the present invention is a very useful invention that can improve the production efficiency of transgenic animals and clones, which is indispensable for in vitro culture of fertilized eggs.

The present invention can improve the production efficiency of transgenic animals and clones, which are indispensable for in vitro culture of fertilized eggs, and can be used in the industry of biotechnology.

1 is a photograph showing that the cell death of blastocyst stage, less apoptosis in the treated group (10 or 100ng / ml recombinant mouse interleukin-6) compared to the control group.

Fig. 2 is a photograph showing that the blastocyst ovulated blastocyst ovum: 100 ng / ml of recombinant mouse interleukin-6 is more ovum than ovulation in the ovum.

Claims (3)

In fertilized and cloned embryos in vitro culture, In vitro culture of mouse fertilized egg characterized in that the addition of recombinant mouse interleukin-6 to the basal medium (M16) culturing the pre-implantation initial embryo and cloned embryo in vitro blastocyst. The method of claim 1, In vitro culture method of the mouse fertilized egg, characterized in that the fertilized egg is a mouse fertilized egg. The method of claim 1, The in vitro culture method of the fertilized mouse, characterized in that the fertilized egg is a mouse clone.

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