An electroporation transfection protection fluid suitable for abalone embryo cell gene editing and application thereof
By using a combination of low ionic strength buffer and cell protectant, the high deformity and mortality rates in abalone electroporation transfection technology were solved, achieving efficient gene editing and transfection results and promoting progress in marine biological research and aquaculture.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- LUDONG UNIVERSITY
- Filing Date
- 2024-12-27
- Publication Date
- 2026-06-26
AI Technical Summary
Existing abalone electroporation transfection technology suffers from low transfection efficiency, high embryo mortality rate, and high malformation rate, making it difficult to meet the needs of efficient gene editing.
A low ionic strength buffer containing NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose, and glutathione, and polyethylene glycol 400 as a cell protectant, is provided as a protective solution for electroporation transfection of abalone embryonic cells, reducing cell damage from electric shock and improving cell survival rate.
It significantly reduced the malformation rate and mortality rate of abalone embryo cells, improved gene transfection and editing efficiency, reduced experimental costs, and promoted the development of marine biological research and aquaculture.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of gene editing technology, and in particular to an electroporation transfection protection solution suitable for gene editing of abalone embryo cells and its application. Background Technology
[0002] Gene editing in mollusks requires suitable high-throughput transfection methods, but currently, there is a lack of high-throughput gene transfection methods suitable for mollusk embryonic cells. Although studies have shown that microinjection can introduce exogenous genes into mollusk embryonic cells, this method has high requirements for microinjection equipment and can only be performed on individual embryos one by one, resulting in low efficiency. Therefore, developing and optimizing high-throughput gene transfection methods suitable for mollusk embryonic cells is particularly important.
[0003] Electroporation is an effective high-throughput gene transfection technique that has been applied to abalone. However, existing abalone electroporation transfection techniques have significant drawbacks, specifically low transfection efficiency or high efficiency accompanied by high embryo mortality and malformation rates. Firstly, regarding transfection efficiency, although electroporation is widely used for various cell types, and commercially available electroporation transfection buffers exist for mammalian cells, they are not suitable for protecting embryonic cells in shellfish electroporation due to significant differences in the organismal composition and living environment compared to mammalian cells. Secondly, a high malformation rate is another important issue. Although electroporation aims to promote the effective transfection of exogenous genes, the electric shock received by embryonic cells during the application of an electric field can damage their cell membranes and internal structures. This damage not only affects normal physiological functions but can also lead to various malformations during subsequent development, such as slow growth, morphological abnormalities, and incomplete organ development, and in severe cases, embryonic cell death. This not only affects the reliability of research results but also negatively impacts the health and survival of the organism. Therefore, improving the efficiency of electroporation transfection technology in shellfish and reducing mortality and malformation rates are among the key challenges in current research. 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 malformation rate. Summary of the Invention
[0004] The purpose of this invention is to provide an electroporation transfection protection solution suitable for gene editing in abalone embryo cells and its application, thereby addressing the problems existing in the prior art. The electroporation transfection protection solution of this invention can significantly reduce the deformity and mortality rates of abalone cells after electroporation, improve gene transfection and gene editing efficiency, and reduce the cost of gene editing experiments. This lays the foundation for accelerating marine biological research and promoting environmental protection, species conservation, and aquaculture.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a protective solution for electroporation transfection of abalone embryo cells, the protective solution comprising 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 this invention is a low-ionic-strength buffer solution, which can reduce the stress and damage to cells during electroporation. Traditional electroporation buffers, such as PBS (phosphate-buffered saline) or other commercially available electroporation solutions, generally have osmotic pressures and ionic strengths suitable for mammalian cells, but not for shellfish cells (such as abalone cells). Shellfish cells require relatively high osmotic pressures in their natural environment; therefore, a low-ionic-strength buffer solution can ensure the physiological state of shellfish cells. Glucose can provide necessary nutritional support for cells and maintain cell viability; glutathione can reduce oxidative damage to cells during electroporation and improve cell survival rate.
[0010] The cell protectant in this invention can enhance cell membrane permeability, reduce the transient irritation of the cell membrane by electroporation, improve cell survival rate after electroporation, reduce the malformation rate, and enhance the efficiency of exogenous gene introduction.
[0011] Optionally, water is used as the solvent, and the electroporation transfection protective solution contains NaCl at a concentration of 150-240 mM, MgSO4 at a concentration of 50-60 mM, CaCl2 at a concentration of 8-12 mM, KCl at a concentration of 7-10 mM, NaHCO3 at a concentration of 1.5-2.0 mM, NaBr at a concentration of 1.0-2.0 mM, K2HPO4 at a concentration of 0.2-0.6 mM, glucose at a concentration of 80-90 mM, glutathione at a concentration of 2-4 mM, and polyethylene glycol 400 at a concentration of 220-260 mM.
[0012] Optionally, using water as a solvent, the electroporation transfection protective solution contains NaCl at a concentration of 200 mM, MgSO4 at a concentration of 54 mM, CaCl2 at a concentration of 10 mM, KCl at a concentration of 8.7 mM, NaHCO3 at a concentration of 1.8 mM, NaBr at a concentration of 1.5 mM, K2HPO4 at a concentration of 0.4 mM, glucose at a concentration of 84 mM, glutathione at a concentration of 3 mM, and polyethylene glycol 400 at a concentration of 250 mM.
[0013] The present invention also provides the application of the above-mentioned electroporation transfection protection solution in the electroporation transfection of abalone cells.
[0014] The present invention also provides an electroporation transfection method for abalone cells, comprising the step of seeding the abalone cells in the above-mentioned electroporation transfection protective solution and performing electroporation transfection.
[0015] Optionally, after inoculation, the density of abalone cells is 80-120 cells / 100μL.
[0016] Optionally, after inoculation, the density of abalone cells is 100 cells / 100 μL.
[0017] Optionally, the abalone cells include wrinkled disc abalone cells.
[0018] Optionally, the wrinkled abalone cells include wrinkled abalone embryonic cells.
[0019] The present invention discloses the following technical effects:
[0020] The electroporation transfection protection solution of this invention can effectively reduce the damage to abalone cells caused by electroporation, thereby reducing the rate of malformations during embryonic development to approximately 19%, and also reducing cell mortality after electroporation to approximately 3%. This electroporation transfection protection solution can help improve the efficiency of gene transfection and gene editing in abalone, accelerating marine biological research, promoting technological advancements in environmental protection, species conservation, and aquaculture, and is of great significance for maintaining marine ecological balance. Furthermore, improving gene transfection and gene editing efficiency can significantly reduce experimental costs and increase research efficiency, enabling related enterprises and research institutions to achieve a higher return on investment when conducting shellfish genetic improvement and related biological research, thus possessing practical application value. Detailed Implementation
[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] Example 1
[0027] 1. Reagent preparation: Anhydrous ethanol: used to clean and disinfect experimental equipment.
[0028] Sterile ultrapure water: the base solvent.
[0029] NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, and glucose: adjust the ionic strength and osmotic pressure of the buffer solution.
[0030] Mannitol: Reduces cell damage caused by electroporation.
[0031] Polyethylene glycol (PEG): Enhances cell membrane permeability.
[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 cell damage caused by electroporation.
[0034] 2. Optimization of electroporation transfection protection solution
[0035] The preparation method of the electroporation transfection protection solution is as follows:
[0036] a. In a sterile environment, add 10 mL of sterile ultrapure water to a beaker;
[0037] b. As needed, gradually add NaCl and other reagents (MgSO4, CaCl2, etc.), mix thoroughly, 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 based on the total prepared volume;
[0040] e. Disinfect 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) Base solution: composed of NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione; see Table 1 for specific formula.
[0043] Table 1 Base Fluid Formulation
[0044]
[0045] Electroporation experiments were used to verify the protective effects of the above-mentioned basic solutions on abalone embryo cells. The experimental procedure is as follows:
[0046] a. Embryonic cell preparation:
[0047] Electroporation experiments were conducted using abalone as a representative example. Three male abalone and twelve female abalone with mature gonads were selected. After stimulating the abalone to release sperm and eggs, the sperm and eggs were mixed for fertilization. The release of polar bodies was observed after 20 minutes. If most egg cells began to release polar bodies, fertilization was considered successful. If only a small number of egg cells released polar bodies, an appropriate amount of sperm was added to ensure fertilization of normal egg cells. After observing polar body release in most egg cells, the fertilized eggs were examined under a microscope every 10 minutes to check for division into two cells. When most fertilized eggs were between two and eight cells, subsequent steps were performed.
[0048] b. Electroporation setup and operation process:
[0049] Fertilized eggs were collected by filtering through a 200-mesh sieve and added to different formulations of basal solutions. The density of fertilized eggs was adjusted to approximately 100 eggs / 100 μL. After gentle mixing, the mixture was transferred to an electroporation tube.
[0050] Set the electroporator parameters, including a voltage of 100V / cm, a pulse duration of 50ms, and a pulse count of 1. Begin electroporation and pause after each pulse to check cell status, controlling the interval time to 30s.
[0051] c. Data statistics:
[0052] After electroporation, the cells were transferred to sterile seawater and placed on a plankton counting plate. Microscopic images were taken for data analysis on malformation and mortality rates. The statistical results are shown in Table 2.
[0053] Table 2. Statistical results of malformation 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 basal solution numbered 2 has the strongest protective effect on the embryonic cells of the wrinkled abalone, reducing the malformation rate to about 26% and the mortality rate to about 6%.
[0056] (2) Cell protectants: including mannitol, polyethylene glycol 400 and bovine serum albumin (BSA); cell protectants are added to the basic solution formula 3, and the specific formula is shown in Table 3.
[0057] Table 3. Base solution + cell protectant formulation
[0058]
[0059] Electroporation was used to verify the protective effect of the above reagents on abalone embryo cells. The experimental procedure is as follows:
[0060] a. Embryonic cell preparation:
[0061] Electroporation experiments were conducted using abalone as a representative example. Three male abalone and twelve female abalone with mature gonads were selected. After stimulating the abalone to release sperm and eggs, the sperm and eggs were mixed for fertilization. The release of polar bodies was observed after 20 minutes. If most egg cells began to release polar bodies, fertilization was considered successful. If only a small number of egg cells released polar bodies, an appropriate amount of sperm was added to ensure fertilization of normal egg cells. After observing polar body release in most egg cells, the fertilized eggs were examined under a microscope every 10 minutes to check for division into two cells. When most fertilized eggs were between two and eight cells, subsequent steps were performed.
[0062] b. Electroporation setup and operation process:
[0063] Fertilized eggs were collected by filtering through a 200-mesh sieve and added to protective solutions with different formulations. The density of fertilized eggs was adjusted to approximately 100 eggs / 100μL. After gentle mixing, the mixture was transferred to an electroporation tube.
[0064] Set the electroporator parameters, including a voltage of 100V / cm, a pulse duration of 50ms, and a pulse count of 1. Begin electroporation and pause after each pulse to check cell status, controlling the interval time to 30s.
[0065] c. Data statistics:
[0066] After electroporation, the cells were transferred to sterile seawater and placed on a plankton counting plate. Microscopic images were taken for data analysis on malformation and mortality rates. The statistical results are shown in Table 4.
[0067] Table 4. Statistical results of malformation 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, cell protectant formulation 5 has the strongest protective effect on the embryonic cells of the wrinkled abalone, reducing the malformation rate to about 25% and the mortality rate to about 22%.
[0070] (3) Combining the results of (1) and (2), reagents numbered 2 and 5 were combined to prepare a protective solution, numbered 8. The protective effect of this protective solution on abalone embryo cells was detected by electroporation, using the wrinkled abalone as a representative. The experimental procedure is as described in (1) and (2). The protective solution formulation and test results are shown in Table 5.
[0071] Table 5. Statistical results of malformation rate and mortality rate
[0072]
[0073] As can be seen from Table 5, the protective solution combining the base solution formula 2 and the cell protectant formula 5 has greatly improved the protective effect on the embryonic cells of the wrinkled abalone, reducing the malformation rate to about 19% and the mortality rate to about 3%.
[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. The application of an electroporation transfection protective solution suitable for gene editing in abalone embryo cells in electroporation transfection of abalone embryo 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; Using water as a solvent, the electroporation transfection protective solution contains the following concentrations: NaCl 200 mM, MgSO4 50-60 mM, CaCl2 8-12 mM, KCl 7-10 mM, NaHCO3 1.5-2.0 mM, NaBr 0.87 mM, K2HPO4 0.2-0.6 mM, glucose 80-90 mM, glutathione 2-4 mM, and polyethylene glycol 400 220-260 mM.
2. The application according to claim 1, characterized in that, Using water as a solvent, the electroporation transfection protective solution contained the following concentrations: NaCl 200 mM, MgSO4 54 mM, CaCl2 10 mM, KCl 8.7 mM, NaHCO3 1.8 mM, NaBr 0.87 mM, K2HPO4 0.4 mM, glucose 84 mM, glutathione 3 mM, and polyethylene glycol 400 250 mM.
3. A method for electroporation transfection of abalone embryonic cells, characterized in that, The procedure includes the step of seeding the abalone embryo cells in the electroporation transfection protection solution described in claim 1 or 2, and performing electroporation transfection.
4. The electroporation transfection method according to claim 3, characterized in that, After inoculation, the density of abalone embryonic cells was 80-120 cells / 100μL.
5. The electroporation transfection method according to claim 4, characterized in that, After inoculation, the density of abalone embryonic cells was 100 cells / 100 μL.
6. The method according to claim 3, characterized in that, The abalone embryo cells are wrinkled disc abalone embryo cells.