A method for introducing exogenous nucleic acid fragments into bivalve sperm
Through the treatment of marine bivalve shellfish sperm, the foreign nucleic acid fragments were successfully introduced, which solved the problems of cumbersome operation, high cost and low efficiency in the existing technology, and achieved efficient and simple plasmid introduction, satisfying shellfish gene editing demand.
Patent Information
- Application Number
- CN202410192006.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-21
- Publication Date
- 2025-02-18
- Estimated Expiration
- 2044-02-21
AI Technical Summary
The prior art has problems such as cumbersome operation, high cost, low efficiency and cytotoxin residue in marine bivalve gene editing, making it difficult to achieve efficient and simple plasmid introduction.
After centrifuging the collected bivalve sperm to remove the supernatant, NaCl solution and CaCl2 solution were added for treatment, and then the exogenous nucleic acid fragment was added and the ice water bath and heat shock treatment was performed to achieve the introduction of the exogenous nucleic acid fragment.
This method can efficiently introduce sgRNA recombinant expression plasmids, with an introduction rate of up to 47% to 70%. It is simple to operate, low cost and short time, and is suitable for batch operations and meets the demand for large egg laying of shellfish.
Smart Images
Figure CN118064501B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene editing, and in particular relates to a method for introducing exogenous nucleic acid fragments into bivalve sperm. Background Art
[0002] Gene editing technology is a direct and effective method for exploring gene functions and analyzing traits in recent years. Among them, CRISPR / Cas9 gene editing technology has the advantages of simple operation, wide target selection, low cost and high efficiency, providing a powerful tool for genetic analysis and breeding of economic traits of important aquatic species. Since 2014, with the increasing maturity of cas9 technology, its application has become more and more extensive. The most popular and widely used CRISPR / Cas9 system is composed of Cas9 protein and sgRNA as the core. Cas9 contains two active sites, RuvC at the amino terminus and HNH in the middle of the protein, which play a role in crRNA maturation and double-stranded DNA shearing, and can cause double-stranded DNA breaks. When the DNA is broken, there are DNA fragments homologous to the damaged DNA in the cell nucleus at the same time, and the exogenous DNA fragments can be introduced at the target site through homology-mediated double-stranded DNA repair, thereby achieving the effect of fragment knock-in or editing. The continuous development of CRISPR / Cas9 technology has brought revolutionary breakthroughs in the research of biology and medicine. The use of this technology to construct gene mutation model organisms is helpful for the study of gene function and has important reference value for the treatment of genetic diseases. It can also effectively improve the production performance of economic animals and enhance their disease resistance at the genomic level.
[0003] Among the gene editing introduction methods of the CRISPR / Cas system, there are three main categories, namely biological transfection (lentivirus, adenovirus, etc.), chemical transfection (liposomes, cationic polymers), and physical transfection (electroporation, microinjection, gene gun). Compared with the above introduction methods, the biological transfection method is relatively easy to operate, but there is a possibility of residual cytotoxins; the most prominent advantage of the chemical transfection method is that it is easy to operate, does not require complex technical requirements, and does not have cytotoxin problems, but its biggest disadvantage is that stability is difficult to guarantee and the transfection efficiency is relatively low; physical transfection greatly reduces the risk of such toxins.
[0004] Marine bivalve molluscs are the main edible aquatic products in my country. Compared with aquatic fish, the application of gene editing breeding technology in shellfish is still in its infancy. At the same time, the small diameter of shellfish eggs, the difficulty of micromanipulation, the high mortality rate of larvae, the long indirect development time, and the difficulty, high cost and long time in obtaining heritable pure lines have also hindered the development of genome editing technology in marine bivalve molluscs.
[0005] In actual operation, due to the small egg diameter of marine bivalve molluscs (including mud clams) and the low survival rate after injection, operations such as microinjection and gene guns are difficult to implement on mud clams. In addition, CN114164232.A discloses a method for electrotransfection gene editing of bivalve molluscs, but the method needs to be operated within the fertilized egg-8 cell stage. After exceeding the 8th stage, the cells are too mature and it is difficult to achieve the editing effect, and the electric field pulse damages the embryo, affects the normal development of the fertilized egg, and has a high teratogenicity rate. At the same time, the electrotransfection method has high requirements for equipment, cumbersome operation, and is not easy to serve the production operations of shellfish breeding. Therefore, it is urgent to have a simple operation, high efficiency, no cytotoxicity, and a batch-operated plasmid introduction method to solve the various problems of mud clams gene editing.
[0006] Sperm-mediated gene transfer utilizes the ability of animal sperm to spontaneously bind and internalize exogenous DNA, and introduces it into the egg cell during fertilization. However, the traditional sperm-mediated method is expensive and cumbersome and is not suitable for aquatic animals, because aquatic organisms are activated by water after sperm release, and after treatment, the sperm motility of aquatic animals is greatly reduced or even causes death. Therefore, the traditional sperm-mediated method cannot effectively introduce exogenous nucleic acid fragments, such as sgRNA, into mud clam sperm. Therefore, constructing a simple and efficient plasmid introduction method has become the primary task in the development of shellfish gene editing. Summary of the invention
[0007] The purpose of the present invention is to provide a method for introducing exogenous nucleic acid fragments into bivalve sperm. In view of the shortcomings of existing shellfish gene editing, such as great difficulty, cumbersome operation, high cost and low efficiency, a method for introducing sgRNA recombinant expression plasmids into bivalve bodies that is efficient, simple, inexpensive, non-cytotoxic and can be operated in batches is provided.
[0008] The method for introducing exogenous nucleic acid fragments into bivalve sperm provided by the present invention comprises the following steps:
[0009] 1) After the collected bivalve mollusc sperm is centrifuged and the supernatant is removed, a 0.9% NaCl solution is added and mixed, and then centrifuged at 3000 rpm and the supernatant is removed;
[0010] 2) adding a 0.01% CaCl2 solution to the precipitate after removing the supernatant in step 1) and mixing by pipetting; then adding the exogenous nucleic acid fragment and placing in an ice water bath;
[0011] The exogenous nucleic acid fragment, as specifically described in the embodiment, is a recombinant plasmid expressing sgRNA;
[0012] 3) Immediately heat shock the solution after the ice bath in step 2) in a 42° C. water bath for 45 seconds;
[0013] 4) The solution after transformation in step 3) is then placed in an ice water bath for 2 minutes to complete the operation of introducing the exogenous fragment into the sperm.
[0014] Experiments have shown that the introduction rate of sgRNA recombinant expression plasmids into the embryos of mud clams after successful fertilization can reach 47% to 70%. Compared with the prior art, the introduction method of the present invention is simple to operate, has low introduction cost, short operation time, can realize batch operation, and meet the actual demand for large spawning of shellfish. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 :tdTomato sgRNA recombinant plasmid map,
[0016] Figure 2 :Expression diagram of tdTomato sgRNA recombinant plasmid in the body of D-type larvae of mud clam,
[0017] Figure 3 :Statistical graph of the introduction rate of tdTomato sgRNA recombinant plasmid. DETAILED DESCRIPTION
[0018] The present invention is further described in detail below with reference to the embodiments.
[0019] Example 1: Transformation and introduction of tdTomato sgRNA recombinant plasmid into mud clams
[0020] Relevant experiments were carried out at the Qingjiang Base of Zhejiang Institute of Marine Fisheries and Zhejiang University in July 2022.
[0021] After the impurities were removed after induced spawning, the sperm of the mud clam was placed in a 0.01% CaCl2 solution for 30 minutes in an ice-water bath, 45 seconds of heat shock, and 2 minutes of ice-water bath for stabilization. Then, the tdTomato sgRNA recombinant expression plasmid was introduced into the sperm of the mud clam in batches, and artificial insemination was successfully completed. After the development to the embryonic stage, the expression status of the recombinant plasmid in the mud clam embryo was observed. The specific operation steps are as follows:
[0022] 1) Plasmid selection: A plasmid vector (lentiCRISPR v2) with Cas9 and tdTomato sequences was used. The cas9 and sgRNA expression frames were on the same vector, and the C-terminus of Cas9 had a FLAG fusion tag. The vector contained a Puromycin resistance gene. After the recombinant plasmid was successfully introduced into the cell, tdTomato was expressed as red fluorescence in the cell, so the tdTomato sgRNA recombinant plasmid was selected as the reporter gene for successful introduction;
[0023] 2) Prepare solution: weigh a certain amount of NaCl into a glass container, add water to prepare a 0.9% NaCl solution; weigh a certain amount of CaCl2 into a glass container, add water to prepare a 0.01% CaCl2 solution;
[0024] 3) Cleaning the mud clams: Use sand-filtered seawater to clean the dirt on the surface of the mud clams;
[0025] 4) Sperm acquisition: After induced spawning of mud clams by simulated environmental stimulation, sufficient high-quality mud clams sperm were collected for transformation. Immediately after the mud clams released sperm, they were collected and centrifuged at 3000 rpm for 3 minutes. After discarding the supernatant (about 100 μL of precipitate), 1 mL of 0.9% NaCl solution was immediately added and mixed. After centrifugation at 3000 rpm for 3 minutes at room temperature, the supernatant was discarded.
[0026] 5) Sperm membrane permeabilization: Add 500 μL of 0.01% CaCl2 solution to the precipitate obtained in step (4) and mix by pipetting immediately. Centrifuge the resulting solution at 3000 rpm for 3 minutes at room temperature, remove the supernatant, and then add 100 μL of 0.01% CaCl2 solution;
[0027] 6) Plasmid attachment: Add 10 μL of sgRNA recombinant expression plasmid (about 2 μg) to the solution obtained in step (5) and place in an ice water bath for 30 minutes (0°C);
[0028] 7) Heat shock conversion: After the ice bath in step (6), the solution was immediately heat-shocked in a 42°C water bath for 45 seconds;
[0029] 8) Sperm stabilization: After the transformation in step (7) is completed, the solution is then placed in an ice water bath for 2 minutes (0°C). The entire operation process is kept stable to reduce shaking and collision;
[0030] Fertilization of mud cockles: Artificial insemination is immediately performed on the mud cockle sperm after transformation in step (8), and purified seawater is replaced regularly.
[0031] After the fertilized mud clam embryos in step (9) develop to the D-type larvae stage, the red fluorescence expression status in the body is observed to determine the expression status of the introduced plasmid.
[0032] Table 1 is a statistical analysis of the transformation results of Example 1. The plasmid transformation was performed three times to reduce technical errors and to have biostatistical significance. After the transformation step was completed, 20 fertilized eggs were taken for each repetition to perform transformation and introduction rate statistics, and the expression of red fluorescence in the embryos after development to D-type larvae was observed and biological statistics were performed.
[0033] Table 1: Plasmid transfer rate statistics
[0034]
[0035] The above results show that the use of the inventive method can efficiently introduce the mud clam sgRNA recombinant plasmid, and the introduction method is simple to operate, with low introduction cost and short operation time, and can realize batch operation to meet the actual needs of large shellfish spawning.
Claims
1. A method for introducing exogenous nucleic acid fragments into bivalve sperm, characterized in that: The method comprises the following steps: 1) After the collected bivalve sperm is centrifuged and the supernatant is removed, NaCl solution is added and mixed, and then centrifuged again and the supernatant is removed; 2) Add CaCl2 solution to the precipitate after removing the supernatant in step 1) and mix well by pipetting, then add the exogenous nucleic acid fragment and place in an ice-water bath at 0°C for 30 minutes; 3) Immediately heat shock the solution after the ice bath in step 2) in a 42°C water bath; 4) The solution after transformation in step 3) is then placed in an ice-water bath at 0°C again to complete the operation of introducing the exogenous fragment into the sperm; The bivalve molluscs are mud clams; The concentration of the CaCl2 solution in step 2) is 0.01%; The heat shock time in step 3) is 45 seconds; The ice water bath time in step 4) is 2 minutes.
2. The method according to claim 1, characterized in that The exogenous nucleic acid fragment is a recombinant plasmid expressing sgRNA.
3. The method according to claim 1, characterized in that The concentration of the NaCl solution in step 1) is 0.9%.
4. The method according to claim 1, characterized in that The centrifugation in step 1) is performed at 3000 rpm.