Electroporation transfection protection liquid suitable for abalone embryonic cell gene editing and application of electroporation transfection protection liquid

Abstract

The invention discloses an electroporation transfection protection liquid suitable for abalone embryonic cell gene editing and application thereof, and belongs to the technical field of gene editing. The electroporation transfection protection liquid comprises a basic liquid and a cell protection agent, the basic liquid is prepared from NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione, and the pH value of the basic liquid is 7-8. The cell protective agent comprises polyethylene glycol 400. The electroporation transfection protection liquid can significantly reduce the malformation rate and death rate of abalone cells after an electroporation technology, improve the gene transfection and gene editing efficiency, reduce the gene editing experiment cost, and lay a foundation for accelerating marine organism research, promoting environmental protection, species conservation and aquaculture.

Description

Technical Field

[0001] The present invention relates to the technical field of gene editing, and in particular to an electroporation transfection protection solution suitable for gene editing of abalone embryonic cells and an application thereof. Background Art

[0002] Gene editing of shellfish requires the use of appropriate high-throughput transfection methods. Currently, there is still a lack of high-throughput gene transfection methods suitable for shellfish embryonic cells. Although studies have shown that microinjection can introduce exogenous genes into shellfish embryonic cells, this method has high requirements for microinjection equipment and can only inject single embryos one by one, which is inefficient. Therefore, it is particularly important to develop and optimize high-throughput gene transfection methods suitable for shellfish embryonic cells.

[0003] Electroporation transfection is an effective high-throughput gene transfection technology, which has been applied to abalone to a certain extent. However, the existing abalone electroporation transfection technology has obvious defects, which are manifested in low transfection efficiency, or high transfection efficiency, but with high embryonic mortality and malformation rates. First, in terms of transfection efficiency, although electroporation is widely used in a variety of cell types, there are mature commercial electroporation transfection buffers for mammalian cells, but due to the large differences in the body composition and living environment of shellfish and mammalian cells, it is not suitable for protecting embryonic cells in shellfish electroporation transfection. Secondly, the high malformation rate is another important issue. Although electroporation technology is designed to promote the effective transfection of exogenous genes, when the electric field is applied, the electric shock to the embryonic cells may cause damage to their cell membranes and internal structures. This damage not only affects the normal physiological function of the cells, but may also lead to various malformations in the subsequent development process, such as slow growth, morphological abnormalities, organ dysplasia, etc., and severe cases can lead to the death of embryonic cells. This not only affects the reliability of the research results, but also has a negative impact on the health and survival of individual organisms. Therefore, improving the efficiency of electroporation transfection technology for shellfish and reducing mortality and deformity rates are one of the key challenges of current research. In order to overcome these obstacles, it is necessary to develop an electroporation buffer or electroporation protection solution specifically for shellfish, especially abalone embryonic cells, to minimize the damage of electric shock to abalone embryonic cells, thereby maintaining a low mortality and deformity rate. Summary of the invention

[0004] The purpose of the present invention is to provide an electroporation transfection protective solution suitable for gene editing of abalone embryonic cells and its application to solve the problems existing in the above-mentioned prior art. The electroporation transfection protective solution of the present invention can significantly reduce the deformity rate and mortality rate of abalone cells after electroporation technology, improve gene transfection and gene editing efficiency, reduce the cost of gene editing experiments, and accelerate marine biological research, promote environmental protection, species conservation and aquaculture. Lay the foundation.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an electroporation transfection protection solution suitable for abalone embryonic cells, wherein the electroporation transfection protection solution comprises a base solution and a cell protectant;

[0007] The base solution contains NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione;

[0008] The cell protectant includes polyethylene glycol 400.

[0009] The base solution in the present invention is a buffer with low ionic strength, which can reduce the pressure and damage to cells during electroporation. The osmotic pressure and ionic strength of traditional electroporation buffers, such as PBS (phosphate buffered saline) or other commercial electroporation solutions, are usually suitable for mammalian cells, but not for shellfish cells (such as abalone cells). Shellfish cells need to maintain a relatively high osmotic pressure in the natural environment, so a buffer with low ionic strength can ensure the physiological state of shellfish cells; glucose can provide cells with necessary nutritional support and maintain cell activity; glutathione can reduce oxidative damage to cells during electroporation and improve cell survival rate.

[0010] The cell protectant of the present invention can enhance the permeability of cell membranes, reduce the transient stimulation of electroporation on cell membranes, improve the survival rate of cells after electroporation, reduce the deformity rate, and enhance the efficiency of introducing foreign genes.

[0011] Optionally, water is used as the solvent, and the concentration of NaCl in the electroporation transfection protection solution is 150-240mM, the concentration of MgSO4 is 50-60mM, the concentration of CaCl2 is 8-12mM, the concentration of KCl is 7-10mM, the concentration of NaHCO3 is 1.5-2.0mM, the concentration of NaBr is 1.0-2.0mM, the concentration of K2HPO4 is 0.2-0.6mM, the concentration of glucose is 80-90mM, the concentration of glutathione is 2-4mM, and the concentration of polyethylene glycol 400 is 220-260mM.

[0012] Optionally, water is used as the solvent, and the concentration of NaCl in the electroporation transfection protection solution is 200mM, the concentration of MgSO4 is 54mM, the concentration of CaCl2 is 10mM, the concentration of KCl is 8.7mM, the concentration of NaHCO3 is 1.8mM, the concentration of NaBr is 1.5mM, the concentration of K2HPO4 is 0.4mM, the concentration of glucose is 84mM, the concentration of glutathione is 3mM, and the concentration of polyethylene glycol 400 is 250mM.

[0013] The present invention also provides an application of the electroporation transfection protection solution in electroporation transfection of abalone cells.

[0014] The present invention also provides an electroporation transfection method for abalone cells, comprising the steps of inoculating the abalone cells into the electroporation transfection protection solution and performing electroporation transfection.

[0015] Optionally, after the inoculation, the density of abalone cells is 80-120 cells / 100 μL.

[0016] Optionally, after the inoculation, the density of abalone cells is 100 cells / 100 μL.

[0017] Optionally, the abalone cells include Haliotis discus henna cells.

[0018] Optionally, the Haliotis discus hannai cells include Haliotis discus hannai embryonic cells.

[0019] The present invention discloses the following technical effects:

[0020] The electroporation transfection protection solution of the present invention can effectively reduce the damage of electroporation to abalone cells, thereby reducing the deformity rate occurring during embryonic development, which can be reduced to about 19%, and can reduce the cell mortality rate after electroporation, which can be reduced to about 3%. The electroporation transfection protection solution of the present invention can help improve the gene transfection and gene editing efficiency of abalone, accelerate marine biological research, promote technological progress in environmental protection, species conservation and aquaculture, and is of great significance to maintaining the balance of marine ecology. In addition, improving the efficiency of gene transfection and gene editing can significantly reduce experimental costs, improve scientific research efficiency, and enable related enterprises and research institutions to obtain a higher rate of return on investment when conducting shellfish genetic improvement and related biological research, which has practical application value. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Example 1

[0027] 1. Reagent preparation: Anhydrous ethanol: for cleaning and disinfecting experimental equipment.

[0028] Sterile ultrapure water: base solvent.

[0029] NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4 and glucose: adjust the ionic strength and osmotic pressure of the buffer.

[0030] Mannitol: Reduces damage to cells caused by electroporation.

[0031] Polyethylene glycol (PEG): enhances the permeability of cell membranes.

[0032] Glutathione: Reduces oxidative damage to cells during electroporation and improves cell survival rate.

[0033] Bovine serum albumin (BSA): It has a protective effect on cell membranes and reduces the damage to cells caused by electroporation.

[0034] 2. Optimization of electroporation transfection protection solution

[0035] The preparation method of electroporation transfection protection solution is as follows:

[0036] a. In a sterile environment, add 10 mL of sterile ultrapure water into a beaker;

[0037] b. As needed, gradually add NaCl and other reagents (MgSO4, CaCl2, etc.), mix well, and ensure complete dissolution;

[0038] c. Add mannitol, polyethylene glycol, BSA or glutathione according to the formula and continue stirring until completely dissolved;

[0039] d. Adjust the volume of the dissolved electroporation transfection protection solution to the target size according to the total volume of the configuration;

[0040] e. Sterilize the equipment with anhydrous ethanol to avoid contamination;

[0041] f. Aliquot the buffer into RNase-free centrifuge tubes, label them clearly and store them at 4°C.

[0042] (1) Basic solution: composed of NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione; the specific formula is shown in Table 1.

[0043] Table 1 Basic liquid formula

[0044]

[0045] Electroporation experiments were used to verify the protective effects of the above-mentioned basal solutions on abalone embryonic cells. The experimental process is as follows:

[0046] a. Embryonic cell preparation:

[0047] The electroporation experiment was carried out with the wrinkled abalone as a representative. Three male abalones and 12 female abalones with mature gonads were selected. After stimulating the abalone to discharge sperm and eggs, the sperm and eggs were mixed for fertilization. After 20 minutes, the polar body discharge was observed. If most of the egg cells began to discharge polar bodies, it means that the fertilization was successful. If only a small number of egg cells discharge polar bodies, add an appropriate amount of sperm so that normal egg cells can be fertilized. After most of the egg cells are observed to discharge polar bodies, check the fertilized eggs under a microscope every 10 minutes to see if they have divided into two cells. When most of the fertilized eggs are between 2 cells and 8 cells, proceed to the subsequent steps.

[0048] b. Electroporation settings and operation process:

[0049] The fertilized eggs were collected by filtering with a 200-mesh sieve, added into the basic solution of different formulas, and the density of the fertilized eggs was adjusted to about 100 / 100 μL. After gently mixing, they were transferred into the electroporation tube.

[0050] Set the parameters of the electroporator, including voltage of 100 V / cm, pulse time of 50 ms, and pulse number of 1. Start electroporation and pause after each electric shock to check the cell status, with a control interval of 30 s.

[0051] c. Statistics:

[0052] After electroporation, the cells were transferred to sterile seawater, placed on a plankton counting plate, and photographed under a microscope for statistical analysis of deformity and mortality rates. The statistical results are shown in Table 2.

[0053] Table 2 Statistical results of deformity rate and mortality rate

[0054] serial number Deformity rate mortality rate 1 46.01％±6.15％ 10.94％±2.25％ 2 26.83％±4.65％ 6.36％±4.55％ 3 61.2％±13.72％ 79.46％±8.12％

[0055] As can be seen from Table 2, the base solution No. 2 has the strongest protective effect on the embryonic cells of Haliotis discus hannai, and the deformity rate can be reduced to about 26%, and the mortality rate can be reduced to about 6%.

[0056] (2) Cytoprotectants: including mannitol, polyethylene glycol 400 and bovine serum albumin (BSA); cell protectants are added to the basic solution formula 3. The specific formula is shown in Table 3.

[0057] Table 3 Basic solution + cell protectant formula

[0058]

[0059] Electroporation experiments were used to verify the protective effects of the above reagents on abalone embryonic cells. The experimental process is as follows:

[0060] a. Embryonic cell preparation:

[0061] The electroporation experiment was carried out with the wrinkled abalone as a representative. Three male abalones and 12 female abalones with mature gonads were selected. After stimulating the abalone to discharge sperm and eggs, the sperm and eggs were mixed for fertilization. After 20 minutes, the polar body discharge was observed. If most of the egg cells began to discharge polar bodies, it means that the fertilization was successful. If only a small number of egg cells discharge polar bodies, add an appropriate amount of sperm so that normal egg cells can be fertilized. After most of the egg cells are observed to discharge polar bodies, check the fertilized eggs under a microscope every 10 minutes to see if they have divided into two cells. When most of the fertilized eggs are between 2 cells and 8 cells, proceed to the subsequent steps.

[0062] b. Electroporation settings and operation process:

[0063] The fertilized eggs were collected by filtering with a 200-mesh sieve, added into protective solutions of different formulations, and the density of the fertilized eggs was adjusted to about 100 eggs / 100 μL. After gently mixing, they were transferred into the electroporation tube.

[0064] Set the parameters of the electroporator, including voltage of 100 V / cm, pulse time of 50 ms, and pulse number of 1. Start electroporation and pause after each electric shock to check the cell status, with a control interval of 30 s.

[0065] c. Statistics:

[0066] After electroporation, the cells were transferred to sterile seawater, placed on a plankton counting plate, and photographed under a microscope for statistical analysis of deformity and mortality rates. The statistical results are shown in Table 4.

[0067] Table 4 Statistical results of deformity rate and mortality rate

[0068] serial number Deformity rate mortality rate 4 43.93％±7.59％ 38.56％±9.44％ 5 25.87％±7.9％ 22.21％±9.69％ 6 29.95％±1.57％ 5.39％±1.94％ 7 34.69％±6.02％ 8.62％±4.2％

[0069] As can be seen from Table 4, the cell protectant formula 5 has the strongest protective effect on the embryonic cells of Haliotis discus hannai, and the deformity rate can be reduced to about 25%, and the mortality rate can be reduced to about 22%.

[0070] (3) Based on the results of (1) and (2), the reagents No. 2 and No. 5 were combined to prepare a protective solution, No. 8, and an electroporation experiment was used to test the protective effect of the protective solution on abalone embryonic cells, with Haliotis discus henna as a representative. The experimental process refers to (1) and (2). The protective solution formula and test results are shown in Table 5.

[0071] Table 5 Statistical results of deformity rate and mortality rate

[0072]

[0073] As can be seen from Table 5, the protective solution obtained by combining the basic solution formula 2 with the cell protectant formula 5 has greatly improved the protective effect on the embryonic cells of the Haliotis discus hannai, with the deformity rate reduced to about 19% and the mortality rate reduced to about 3%.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An electroporation transfection protection solution suitable for gene editing of abalone embryonic cells, characterized in that: The electroporation transfection protection solution includes a base solution and a cell protectant; The base solution contains NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione; The cell protectant includes polyethylene glycol 400.

2. The electroporation transfection protection solution according to claim 1, characterized in that With water as solvent, the concentration of NaCl in the electroporation transfection protection solution is 150-240mM, the concentration of MgSO4 is 50-60mM, the concentration of CaCl2 is 8-12mM, the concentration of KCl is 7-10mM, the concentration of NaHCO3 is 1.5-2.0mM, the concentration of NaBr is 1.0-2.0mM, the concentration of K2HPO4 is 0.2-0.6mM, the concentration of glucose is 80-90mM, the concentration of glutathione is 2-4mM, and the concentration of polyethylene glycol 400 is 220-260mM.

3. The electroporation transfection protection solution according to claim 2, characterized in that With water as solvent, the concentration of NaCl in the electroporation transfection protection solution is 200mM, the concentration of MgSO4 is 54mM, the concentration of CaCl2 is 10mM, the concentration of KCl is 8.7mM, the concentration of NaHCO3 is 1.8mM, the concentration of NaBr is 1.5mM, the concentration of K2HPO4 is 0.4mM, the concentration of glucose is 84mM, the concentration of glutathione is 3mM, and the concentration of polyethylene glycol 400 is 250mM.

4. Use of the electroporation transfection protection solution according to any one of claims 1 to 3 in abalone cell electroporation transfection.

5. An electroporation transfection method for abalone cells, characterized in that: The method comprises the steps of inoculating the abalone cells into the electroporation transfection protection solution according to any one of claims 1 to 3, and performing electroporation transfection.

6. The electroporation transfection method according to claim 5, characterized in that: After the inoculation, the density of abalone cells is 80-120 cells / 100 μL.

7. The electroporation transfection method according to claim 6, characterized in that: After the inoculation, the density of abalone cells was 100 cells / 100 μL.

8. The method according to claim 5, characterized in that The abalone cells include Haliotis discus hannai cells.

9. The method according to claim 8, characterized in that The Haliotis discus hannai cells include Haliotis discus hannai embryonic cells.

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