Electro-transfection buffer solution suitable for long-fragment mRNA transfection cells as well as preparation method and application of electro-transfection buffer solution

Abstract

The invention discloses an electrotransfection buffer solution suitable for long-fragment mRNA transfection cells as well as a preparation method and application of the electrotransfection buffer solution. The electrotransfection buffer solution is prepared from the following components with final concentration: 100 to 150mmol / L of sodium chloride, 1 to 5mmol / L of potassium chloride, 5 to 15mmol / L of disodium hydrogen phosphate, 1 to 3mmol / L of monopotassium phosphate, 0.001 to 0.2 mmol / L of glucose, 1 to 15mmol / L of magnesium chloride, 0.1 to 1.0 mmol / L of calcium chloride and the balance of water. The electroporation buffer solution is low in permeability and conductivity, and high electroporation efficiency can be achieved; through comparison, the transfection efficiency of the electrotransfection buffer solution is obviously higher than that of a commercial electrotransfection buffer solution and a complete culture solution under the condition of the same dosage, and the electrotransfection buffer solution has practical application value.

Description

Technical Field

[0001] The present invention specifically relates to an electroporation buffer suitable for transfection of long - fragment mRNA into cells, a preparation method thereof and an application thereof. Background Art

[0002] Electroporation is a method of introducing polar molecules into host cells by mechanical means. Its principle is that after the electric field acts on cells for several microseconds to several milliseconds, small pores or openings are temporarily formed on the cell membrane, and macromolecules (such as nucleic acids RNA, DNA, etc.) are introduced into the cells and finally enter the cell nucleus. The electroporation technology can not only transfer DNA, RNA, but also transfer antibodies, enzymes and other bioactive molecules into bacteria, yeast, animal cells and plant cells. It is an efficient and simple gene transfer system, with advantages unparalleled by other transfer methods, such as simple and fast operation, strong repeatability, high transfection rate, low price, wide application spectrum, etc. Especially for suspension - cultured cells that are generally considered difficult to transfect at present, a relatively high transfection rate can also be obtained. This technology has become a quite effective non - viral gene transfer technology and is widely used in biological research and the medical field.

[0003] At the present stage, common electroporation buffers can be divided into the following three categories in terms of their composition: The first category is cell culture media, such as RPMI1640, DMEM, DMEM / F12, Opti - MEM, etc.; the second category is phosphate buffers, such as K - PBS, HBS, HeBs, PBS, etc.; the third category is Cytomix buffer, and the Cytomix formula is as follows: 120 mmol / L KCl, 0.15 mmol / L CaCl2, 10 mmol / L K2HPO4 (pH = 7.6), 25 mmol / L HEPES (pH = 7.6), 2 mmol / L EGTA (pH = 7.6), 5 mmol / L MgCl2, 2 mmol / L ATP, 5 mmol / L glutathione. In terms of composition, the first category uses cell culture media as electroporation buffers, with relatively complex components, which is not conducive to the stability of mRNA transfection. The second category uses phosphate buffers as electroporation buffers, which are applied to mRNA transfection, but are not conducive to the transfection of long - fragment mRNA. The third category is mainly Cytomix buffer or modified buffers, which belong to high - K+ buffers. It is precisely because of the too - high potassium ion concentration that increases the conductivity of the whole solution, which is extremely likely to cause damage to cells under high - voltage conditions, destroying cell morphology and viability. In addition, the too - high potassium ion concentration results in the osmotic pressure of the buffer being higher than that in the cells, which is also not conducive to the entry of exogenous mRNA. When attempting to use existing commercial buffers for long - fragment mRNA transfection, there will be a phenomenon of high cell mortality, or very low cell transfection efficiency, or both situations occurring simultaneously. Summary of the Invention

[0004] To solve the above problems, the present invention provides an electroporation buffer suitable for transfecting cells with long - fragment mRNA, which is composed of components with the following final concentrations:

[0005] Sodium chloride 100 - 150 mmol / L, potassium chloride 1 - 5 mmol / L, disodium hydrogen phosphate 5 - 15 mmol / L, potassium dihydrogen phosphate 1 - 3 mmol / L, glucose 0.001 - 0.2 mmol / L, magnesium chloride 1 - 15 mmol / L, calcium chloride 0.1 - 1.0 mmol / L, and the rest is water.

[0006] Furthermore, it is composed of components with the following final concentrations:

[0007] Sodium chloride 136 - 138 mmol / L, potassium chloride 2 - 4 mmol / L, disodium hydrogen phosphate 9 - 11 mmol / L, potassium dihydrogen phosphate 1 - 3 mmol / L, glucose 0.05 - 0.07 mmol / L, magnesium chloride 9 - 11 mmol / L, calcium chloride 0.3 - 0.5 mmol / L, and the rest is water.

[0008] Even further, it is composed of components with the following final concentrations:

[0009] Sodium chloride 136 - 137 mmol / L, potassium chloride 3 - 4 mmol / L, disodium hydrogen phosphate 10 - 11 mmol / L, potassium dihydrogen phosphate 1 - 2 mmol / L, glucose 0.05 - 0.06 mmol / L, magnesium chloride 9 - 10 mmol / L, calcium chloride 0.3 - 0.4 mmol / L, and the rest is water.

[0010] Even further, it is composed of components with the following final concentrations:

[0011] Sodium chloride 136.75 mmol / L, potassium chloride 2.68 mmol / L, disodium hydrogen phosphate 10.1 mmol / L, potassium dihydrogen phosphate 1.76 mmol / L, glucose 0.056 mmol / L, magnesium chloride 10.00 mmol / L, calcium chloride 0.30 mmol / L, and the rest is water.

[0012] Even further, the pH value of the electroporation buffer is 7.1 - 7.7.

[0013] Even further, the conductivity of the electroporation buffer is 14.0 - 15.0 ms / cm, and the osmotic pressure is 200 - 450 mOsm / Kg.

[0014] The present invention also provides a method for preparing the aforementioned electroporation buffer, which includes the following steps:

[0015] Weigh the components according to the ratio, dissolve them in water, adjust the pH value of the dissolution solution to 7.1 - 7.7, and filter and sterilize to obtain the product.

[0016] Further, the water is deionized double-distilled water or RNase-free water.

[0017] The present invention provides the use of the aforementioned electroporation buffer in RNA transfection of cells.

[0018] Further, the RNA includes long fragment mRNA.

[0019] Further, the long fragment mRNA includes CMV-VEEV-EGFP, CMV-VEEV-helper C saRNA, and / or CMV-VEEV-helper E saRNA.

[0020] Further, the cells include BHK-21 cells.

[0021] The saRNA of the present invention is a long fragment mRNA with self-amplifying ability.

[0022] The long fragment mRNA of the present invention refers to mRNA with a nucleotide sequence > 2000 nt.

[0023] The beneficial effects of the present invention:

[0024] 1. The electroporation buffer prepared by the present invention has obvious advantages in cost. The price of commercially available electroporation buffer is relatively high. The value of 100 ml of commercially available electroporation buffer is about 300 - 1500 yuan, while the electroporation buffer of the present invention is composed of ordinary electrolytes, osmotic pressure regulators, and sugars, and the value of each 100 ml is less than 1 yuan, with low cost in actual application.

[0025] 2. The electroporation buffer of the present invention can be used in combination with mainstream electroporators, providing a buffer for electroporation that can simultaneously achieve high cell survival rate and high transfection rate, and is suitable for the vast majority of eukaryotic cells and some prokaryotic cells.

[0026] 3. The electroporation buffer of the present invention has low osmolarity and low conductivity, and can achieve high electroporation efficiency; by comparing the transfection efficiency of traditional commercial electroporation buffer and complete culture medium, the transfection efficiency of the electroporation buffer of the present invention is significantly higher than that of commercial electroporation buffer and complete culture medium under the same dosage.

[0027] 4. During the process of transfecting cells with long-chain mRNA using the electroporation buffer of the present invention, the cell properties do not change significantly: the morphology is intact, the viability is high, and the expression level of exogenous mRNA is significantly higher. It is suitable for transfection buffer of single mRNA or multiple mRNAs of all cells. The transfected macromolecules are directly dissolved in the transfection buffer without any other transfection reagents.

[0028] Obviously, based on the above content of the present invention, and in accordance with the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0029] The following is a further detailed description of the above content of the present invention by means of specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0030] Figure 1 Comparison of positive transfection rates (%) of different buffers;

[0031] Figure 2 Comparison of cell survival rates (%) of different buffers;

[0032] Figure 3 Comparison of fluorescence observations of cells co-transfected with multiple mRNAs mediated by different buffers (Note: VEE-nsp1-519C / E refers to the packaging plasmids CMV-VEEV-helper C saRNA and CMV-VEEV-helper E saRNA). Detailed Description of the Invention

[0033] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all obtained by purchasing commercially.

[0034] Example 1 Preparation of Electroporation Buffer Suitable for Transfecting Cells with Long-Fragment mRNA in the Present Invention

[0035] Formula: 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.01 g of glucose, 0.95 g of magnesium chloride, 0.0333 g of calcium chloride;

[0036] Preparation method: Weigh the raw materials according to the formula, dissolve them in 1000 mL of double-distilled water, adjust the pH to 7.4 with NaOH solution, and filter and sterilize with a filter to obtain the electroporation buffer;

[0037] The final concentrations of the components in the prepared electroporation buffer are:

[0038]

[0039]

[0040] The beneficial effects of the present invention are further illustrated by the following experimental examples.

[0041] Experimental Example 1: Study on Electroporation Buffer for Co-Transfection of Multiple Long-Fragment mRNAs

[0042] 1. Screening of Formulation and Dosage of Electroporation Buffer

[0043] The components in the electroporation buffer have various effects on the electroporation effect, involving multiple factors such as osmotic pressure, pH value, and ion concentration. These components significantly change the electroporation effect by affecting the stability of the cell membrane, the transfer efficiency of nucleic acids, and the cell survival rate. In previous experiments, for the target long-fragment RNA of electroporation, it was determined that the electroporation buffer composed of electrolyte components (sodium chloride, potassium chloride, magnesium chloride, and calcium chloride), osmotic pressure regulators (disodium hydrogen phosphate, potassium dihydrogen phosphate), and sugars (glucose) is beneficial for its electroporation. On this basis, the dosage ratio was further adjusted to obtain an electroporation buffer suitable for co-transfection of multiple long-fragment mRNAs.

[0044] Prepare three groups of buffers with different concentrations of sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate (A1, A2, A3) and a mixture of glucose, magnesium chloride, and calcium chloride (B1, B2, B3) according to Table 1; then combine them to form the final electroporation buffer.

[0045] Table 1 Composition of Self-Prepared Electroporation Buffer SGPX

[0046]

[0047] Use different electroporation buffers to mediate the transfection of BHK-21 cells with the relatively long fragment EGFP mRNA, namely CMV-VEEV-EGFP. Since CMV-VEEV-EGFP carries EGFP (Enhanced Green Fluorescent Protein), if it is successfully transfected into BHK-21 cells, the transfected cells can be observed to have specific green fluorescence under an inverted fluorescence microscope. The higher the percentage of fluorescent cells in the total number of cells, the higher the transfection efficiency. At 24 h - 48 h after electroporation, digest the adherent BHK-21 cells with trypsin, collect the cells by centrifugation, wash them twice with PBS, and then use a flow cytometer to sort the cells with green fluorescence and count the transfection efficiency. The applicability of the electroporation buffer was judged by the transfection efficiency. The results are shown in Table 2.

[0048] Table 2 Transfection Efficiency of Self-Prepared Electroporation Buffer SGPX Mediated EGFP Transfection (%)

[0049]

[0050] Note: Transfection efficiency = number of green fluorescent cells / total number of cells × 100%

[0051] As can be seen from Table 2, even when the types of components in the electroporation buffer are the same, changes in the proportioning can also affect the transfection efficiency of mRNA. The main influencing factor is Component A, namely sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate. The secondary factor is Component B, namely glucose, magnesium chloride, and calcium chloride. When the component proportioning is A2B2 and A2B3, the transfection efficiency is greater than 90%. Among them, the transfection efficiency using A2B2 is the highest at 93.74%. Therefore, the electroporation buffer with the A2B2 proportioning was used for further experiments.

[0052] 2. Verification of the functions of different electroporation buffers

[0053] 2.1 Experimental electroporation buffers

[0054] The electroporation buffer (SGPX) of the present invention: Take 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.01 g of glucose, 0.95 g of magnesium chloride, and 0.0333 g of calcium chloride, and fully dissolve them in 1000 mL of double-distilled water. Adjust the pH to 7.4 with NaOH solution, and filter and sterilize with a filter to obtain it; the final concentrations of each component in the prepared electroporation buffer are:

[0055] Component Final concentration / mmol / L Sodium chloride 136.75 Potassium chloride 2.68 Disodium hydrogen phosphate 10.1 Potassium dihydrogen phosphate 1.76 Glucose 0.056 Magnesium chloride 10.00 Calcium chloride 0.30

[0056] Cytomix T4 electroporation buffer: Purchased from BTX Company.

[0057] SKGA electroporation buffer: Purchased from Yida Biotechnology Co., Ltd.

[0058] Self-made buffer + Cytomix buffer: Take the electroporation buffer of the present invention and Cytomix T4 electroporation buffer, and mix them in a volume ratio of 1:1;

[0059] Self-made buffer + SKGA buffer: Take the electroporation buffer of the present invention and SKGA electroporation buffer, and mix them in a volume ratio of 1:1;

[0060] RPMI1640 electroporation buffer: Purchased from Gibco Company.

[0061] 2.2 Determination of the physicochemical properties of different electroporation buffers

[0062] Take each electroporation buffer, observe its appearance, and measure its conductivity. The results are shown in Table 3.

[0063] Table 3 Comparison of the conductivities of different electroporation buffers

[0064]

[0065] As can be seen from Table 3, the conductivity of the electroporation buffer of the present invention is higher than that of the currently commercially available electroporation buffers. Since cells are prone to breakage due to the osmotic pressure difference in buffers with low conductivity, causing cell injury, while a higher conductivity is beneficial for cells to maintain a normal state and does not damage the structure of long-fragment mRNA, it is therefore more suitable for long-fragment mRNA transfection.

[0066] 2.3 Evaluation of the electroporation effect of different electroporation buffers

[0067] CMV-VEEV-EGFP is the EGFP mRNA based on the Venezuelan equine encephalitis virus system. Acting in synergy with the defective packaging plasmids CMV-VEEV-helper C and CMV-VEEV-helper E saRNA, which are also mRNA, it can package replication-defective virus infection particles (VRP viruses) with transient infectivity. Therefore, the electroporation effect of the electroporation buffer is evaluated by the positive transfection rate of cells, cell viability, and VRP virus titer during the preparation of VRP viruses.

[0068] 2.3.1. Method

[0069] (1) Digest cells: Aspirate the adherent cell culture medium of BHK-21 cells cultured in a T225 culture flask, wash with a small amount of PBS and then aspirate the PBS, and add 3 mL of 0.25% trypsin to digest the cells for 1 - 2 min;

[0070] (2) Terminate digestion: Add a sufficient amount of complete culture medium to terminate digestion.

[0071] (3) Collect cells and count: Take the BHK-21 cells in the logarithmic growth phase, suspend them evenly and place them in an EP tube, and perform counting by trypan blue staining;

[0072] (4) Centrifuge: Transfer the required culture medium cells to a new centrifuge tube, centrifuge at 1000 rpm for 5 min;

[0073] (5) Wash with PBS: Discard the supernatant culture medium to obtain the required cells, add an appropriate amount of PBS to resuspend the cells, and centrifuge at 1000 rpm for 5 min;

[0074] (6) Electroporate: Add the cell suspension mixed with mRNA into the electroporation cuvette at 100 μL / well, insert the electroporation cuvette into the base, and select the correct electroporation conditions for electroporation;

[0075] Note: Take out the electroporation buffer, cell culture medium, and PBS 30 min before electroporation and let them return to room temperature in advance. There should be no air bubbles in the suspension formed by mixing EGFP mRNA, packaging plasmid, cells, and electroporation buffer. If there are air bubbles, they need to be removed.

[0076] (7) Cultivation: Incubate at room temperature for 5 - 10 min after electroporation. Add 200 μL of maintenance medium to the electroporation cup after electroporation, and then transfer it to a T25 culture flask pre - added with 5 mL of DMEM maintenance medium for culture at 37 °C in an O₂ incubator. After transfection, culture it directly in a 37 °C, 5% CO₂ incubator until the target gene is expressed.

[0077] (8) Detection: Observe the fluorescence expression of cells under a fluorescence microscope 24 - 48 h later. Count the proportion of fluorescent cells (positive transfection rate) and cell viability.

[0078] Note: The level of the positive transfection rate is roughly calculated by photographing under a fluorescence microscope and recording the percentage of green - fluorescent cells in the total number of cells, and accurately calculated by sorting green - fluorescent cells using a flow cytometer after trypsin - digesting adherent cells. Cell viability can be calculated by trypsin - digesting adherent cells and using the property that trypan blue can stain dead cells blue while live cells cannot, combined with a cell counter (such as a Countstar automatic cell counter) to count the percentage of live cell numbers.

[0079] (9) Collect the culture supernatant of cells after electroporation, co - incubate with freshly seeded BHK - 21 cells, observe the green - fluorescence - carrying situation of cells under a fluorescence microscope 24 - 48 h later, and determine the VRP titer according to the following steps:

[0080] a: Collect the cell culture supernatant at 24 h respectively, store it at - 80 °C for standby.

[0081] b: Sample dilution: Prepare sterilized 1.5 ml EP tubes according to the number of samples, arrange them in a row from left to right on a tube rack, and add 900 μl of DMEM culture medium (containing 2% newborn bovine serum and double antibodies) to each tube. Take 100 μl of the sample to be tested for 10 - fold serial dilution, and the dilution factors are 10¹ - 10⁻⁸.

[0082] c: Virus inoculation: Discard the supernatant of the 96 - well plate, and add 100 μl of DMEM culture medium containing 2% newborn bovine serum and double antibodies to each well. Select an appropriate dilution factor to inoculate cells, inoculate 6 wells for each dilution factor, inoculate 0.1 ml per well, and set up negative control (DMEM culture medium containing 2% newborn bovine serum and double antibodies) and positive control at the same time. At this time, the final volume per well is 200 μl. Place the 96 - well plate in a 37.0 °C, 5% CO₂ incubator for continuous culture for 3 - 5 days, and observe and count the number of cytopathic effects.

[0083] d: Virus content calculation: Calculate the virus content of the sample to be tested by the Reed - Muench method (TCID 50 ).

[0084] Test establishment conditions: Obvious cytopathic effects (cell rounding, death, detachment, etc.) appear in the positive control wells, and no specific cytopathic effects appear in the negative control wells.

[0085] 2.3.2. Results

[0086] The results of the positive transfection rate of cells electrotransfected with different electrotransfection buffers are shown in Figure 1 , the cell survival rate is shown in Figure 2 , and the fluorescence observation results are shown in Figure 3 . The titers of VRP virus particles packaged by electrotransfected cells are shown in Table 4.

[0087] Table 4 Results of VRP virus titers of cells co-electrotransfected with multiple mRNAs mediated by different electrotransfection buffers

[0088]

[0089] It can be seen from Figures 1 - 3 that: Different electrotransfection buffers have different electrotransfection effects on cells; The Cytomix electrotransfection buffer can only ensure a high electrotransfection efficiency, and the RPMI1640 electrotransfection buffer can only ensure a high cell survival rate; Only under the action of the electrotransfection buffer of the present invention, both the electrotransfection efficiency and the cell survival rate of cells are relatively high.

[0090] CMV-VEEV-EGFP, CMV-VEEV-helper CsaRNA, and CMV-VEEV-helper EsaRNA together constitute a replication-defective Venezuelan equine encephalitis virus expression vector system. Among them, CMV-VEEV-EGFP is a structural protein replicon, and a foreign gene EGFP is added to the structural protein region after the promoter. CMV-VEEV-helper C saRNA and CMV-VEEV-helper EsaRNA are virus replication helpers, and a part of the non-structural protein region is artificially deleted in both of them. When the above 3 saRNAs are co-transfected into host cells (here refers to BHK-21 cells), the packaged virus particles can only infect host cells once, and their progeny viruses cannot self-replicate, resulting in the recombinant virus particles showing the characteristics of replication defect. Therefore, the titer of virus particles is positively correlated with the number of SaRNAs transfected into host cells. The higher the virus particle titer, the more SaRNAs are transfected into cells, and the higher the electrotransfection efficiency. It can be seen from Table 4 that: Compared with commercially available electrotransfection buffers and mixed electrotransfection buffers composed of commercially available electrotransfection buffers and the electrotransfection buffer of the present invention, the titer of virus particles prepared by the action of the electrotransfection buffer of the present invention is the highest.

[0091] In summary, the electroporation buffer of the present invention has a higher transfection efficiency compared to traditional commercial electroporation buffers and complete culture media; during the transfection of long-chain mRNA into cells, it ensures that the cell properties do not change significantly, the cell morphology is intact, and the cell viability is high; the expression level of exogenous mRNA is significantly higher, and it is suitable for the transfection of single or multiple mRNAs into cells, thus having practical value for popularization and application.

Claims

1. An electroporation buffer suitable for transfecting cells with long - fragment mRNA, characterized in that: It is composed of components with the following final concentrations: Sodium chloride 100 - 150 mmol / L, potassium chloride 1 - 5 mmol / L, disodium hydrogen phosphate 5 - 15 mmol / L, potassium dihydrogen phosphate 1 - 3 mmol / L, glucose 0.001 - 0.2 mmol / L, magnesium chloride 1 - 15 mmol / L, calcium chloride 0.1 - 1.0 mmol / L, and the rest is water.

2. The electroporation buffer according to claim 1, wherein: It is composed of components with the following final concentrations: Sodium chloride 136 - 138 mmol / L, potassium chloride 2 - 4 mmol / L, disodium hydrogen phosphate 9 - 11 mmol / L, potassium dihydrogen phosphate 1 - 3 mmol / L, glucose 0.05 - 0.07 mmol / L, magnesium chloride 9 - 11 mmol / L, calcium chloride 0.3 - 0.5 mmol / L, and the rest is water.

3. The electrotransfer buffer according to claim 1 or 2, characterized in that: The pH value of the electroporation buffer is 7.1 - 7.

7.

4. The electroporation buffer according to claim 3, wherein: The conductivity of the electroporation buffer is 14.0 - 15.0 ms / cm, and the osmotic pressure is 200 - 450 mOsm / Kg.

5. A method for preparing the electroporation buffer according to any one of claims 1 to 4, characterized in that: It includes the following steps: Weigh the components according to the ratio, dissolve them in water, adjust the pH value of the dissolution solution to 7.1 - 7.7, and filter and sterilize to obtain it.

6. The method according to claim 5, characterized in that: The water is deionized double-distilled water or RNase-free water.

7. Use of the electroporation buffer according to any one of claims 1 to 4 in RNA transfection of cells.

8. The use according to claim 7, wherein: The RNA includes long fragment mRNA.

9. The use according to claim 7, characterized in that: The long fragment mRNA includes CMV-VEEV-EGFP, CMV-VEEV-helper CsaRNA, and / or CMV-VEEV-helper EsaRNA.

10. The use according to claim 7, wherein: The cells include BHK-21 cells.

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