CN113930449A - In-situ plasmid transfection method for drosophila larva tissue and organs - Google Patents

In-situ plasmid transfection method for drosophila larva tissue and organs Download PDF

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CN113930449A
CN113930449A CN202111111905.2A CN202111111905A CN113930449A CN 113930449 A CN113930449 A CN 113930449A CN 202111111905 A CN202111111905 A CN 202111111905A CN 113930449 A CN113930449 A CN 113930449A
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CN113930449B (en
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张岩
叶晓蕾
冯颖
林益
林雪
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Wenzhou Medical University
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Abstract

The invention discloses an in-situ plasmid transfection method for drosophila larva tissue organs, and relates to the technical field of biology. The method comprises the following steps: s1 preparing tissue and organs to be transfected by drosophila larvae; s2 transfection: transferring the fruit fly tissue organ into a 96-hole culture dish, and performing transfection on the incubation transfection mixed solution A, stable transfection of an incubation pre-culture solution B and conventional culture of the tissue organ after transfection by using a full culture solution C in sequence; s3 transfection assay: and (5) harvesting the tissues and organs transfected by the step S2, fixing by formaldehyde, and observing the transfection effect by adopting conventional immunofluorescence staining. The method can directly transfect the drosophila larva tissue organ, can observe that the drosophila tissue organ realizes the transfection and successful expression of the exogenous gene within 48 hours, and is suitable for the operation environment with bacteria because the technical scheme implements the rule of changing liquid within 12 hours and the cultivation of the drosophila tissue organ is 25 ℃, so the proliferation speed of the microbial contamination is far less than the pollution degree.

Description

In-situ plasmid transfection method for drosophila larva tissue and organs
Technical Field
The invention relates to the technical field of biology, in particular to an in-situ plasmid transfection method for drosophila larvae tissue organs.
Background
Transfection (transfection) is the process of actively or passively introducing foreign DNA or RNA fragments into cells in vitro or in vivo by appropriate biotechnological treatment, followed by protein expression or acquisition of a new phenotype (e.g., RNAi) in the cells. Conventional transfection techniques can be divided into transient transfection, in which the foreign DNA/RNA is not integrated into the host chromosome and multiple copies of nucleic acid are present in the host cell at the same time, so that the expression level is high and can usually only be maintained for several days, and stable transfection, in which the foreign DNA is either integrated into the host chromosome or is present as an episome.
Modern transfection methods are divided into: chemical, physical and viral transfection methods. Wherein the chemical method comprises: calcium phosphate method, DEAE-dextran method, liposome method, etc.; the physical method comprises the following steps: microinjection, electroporation, gene gun, and the like; the calcium phosphate removal method by a chemical method is characterized in that other types of the method need commercial transfection reagents, the reagents have high price and large dosage, so that the cost of single transfection is high, and in addition, the commercial transfection reagents can only transfect cell-level samples and are ineffective to tissues; the physical principle requires expensive instruments such as a microinjection instrument, an electrotransfection instrument, a gene gun and the like; the virus transfection method needs commercial virus or self-packaging virus, the operation flow is complicated, and the existing adenovirus transfection vectors and the like are ineffective to drosophila cell tissues.
Meanwhile, in the prior art, the conventional transfection process is generally only operated on a cell-level sample in a growth period, so a high-standard sterile cell culture environment is required, and mutant cell strains with poor proliferation and division capabilities and tissue masses with large volumes are hardly effective, thereby further increasing the transfection cost.
Disclosure of Invention
The invention provides an in-situ plasmid transfection method for drosophila larva tissue organs, and aims to solve the problems in the background technology.
In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:
an in-situ plasmid transfection method for drosophila larva tissue organs comprises the following steps:
s1 preparation of Drosophila larvae tissue organs: taking a plurality of fruit fly larvae as a group, repeatedly cleaning the fruit fly larvae in PBST for several times to remove dirt and pollutants outside the body surface, then dripping a sterilized PBS cleaning solution into a culture dish, dissecting the fruit fly larvae in the cleaning solution, and avoiding damaging intestinal tracts as much as possible to cause the pollution caused by the outflow of contents, and intercepting the upper sections of the fruit fly larvae, including eyes, wings, legs, balance bars, brains, central nervous systems, salivary glands and epidermis attached to the eyes, the wings, the legs, the balance bars, the brains and the central nervous systems;
the Drosophila three-instar larvae under common feeding conditions can be used in the experiment, and the accidental pollution probability can be reduced by feeding the Drosophila three-instar larvae under sterile or bacteria-reducing conditions.
S2 transfection: transferring the fruit fly tissue organ to a 96-hole culture dish, adding the transfection mixed solution A, and transfecting on a shaker at a slow speed; completely sucking the transfection mixed solution A, quickly cleaning the transfection mixed solution A by using PBS, adding the pre-culture solution B, and culturing for 4-6 hours at 25 ℃; replacing the pre-culture solution B with a full culture solution C for culture; generally, less than 5 larvae tissue organs can be cultured and transfected in each well;
s3 transfection assay: harvesting the tissue and organ transfected by the step S2, fixing the tissue and organ with formaldehyde, and observing the transfection effect by adopting conventional immunofluorescence staining;
the preparation method of the transfection mixed solution A comprises the following steps: adding 1ul of 5% digitoxin concentrated stock solution and 200ng of plasmid to be transfected into 100ul of transfection mixed liquor mother liquor, wherein the main body of the transfection mixed liquor mother liquor is SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM, 10ul of 200mM glutamine, 2ul of 60% sodium lactate and 10ul of 100mM sodium pyruvate are added into 1ml of the culture MEDIUM, and 1ug of pepsin inhibitor, 10ug of aprotinin and 1ug of leupeptin are also added into 1ml of the transfection mixed liquor mother liquor;
subpackaging insect culture medium into 1ml, storing with sodium pyruvate at-20 deg.C, and dissolving to room temperature before use; the glutamine and three protease inhibitor concentrated solutions are frozen and stored at-80 ℃, a small amount of glutamine and three protease inhibitor concentrated solutions are stored to-20 ℃ when being temporarily used, the glutamine and three protease inhibitor concentrated solutions are used within one month, and the glutamine and three protease inhibitor concentrated solutions are discarded after expiration; storing 60% sodium lactate stock solution at 4 ℃;
the digitonin concentrated stock solution is stored at-80 deg.C for 3 months at-20 deg.C;
the preparation method of the preculture solution B comprises the following steps: adding 200ng of plasmid to be transfected into 100ul SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM;
the preparation method of the whole culture solution C comprises the following steps: the culture MEDIUM was added to SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM, wherein 10ul of 200mM glutamine, 2ul of 60% sodium lactate, 10ul of 100mM sodium pyruvate, 10ul of 100X (preferably at a defined concentration, which is not allowed to have this vague idea) penicillin and streptomycin double anti-concentrated stock solution and 100ul of fetal bovine serum were added to 900ul of the MEDIUM.
Placing the whole culture solution in a refrigerator at 4 deg.C, balancing to room temperature before use, and refrigerating.
In the present invention, in step S1, the number of the larvae of drosophila per group is not more than 5, the surface of the larvae of drosophila is washed repeatedly with PBST 3 times, and dissected in a culture dish containing sterilized PBS.
In step S2, after dissection, the drosophila tissue and organ were rapidly washed twice with PBS, and each washing process was as follows: after 10 dropwise washes with PBS wash and every two washes and transfers of tissue organs, the forceps were flame sterilized and cooled in air for use.
The specific operation can be as follows: dripping 10 drops of PBS (phosphate buffer solution) into a culture dish, wherein each drop is about 60-80ul, after dissection is completed, washing each tissue organ of the drosophila larvae drop by drop, so that possible pollutants on the surfaces of each tissue organ and in gaps of the drosophila larvae are washed as much as possible, sterilizing and cooling the operation by using forceps alcohol lamp flame, and using the forceps once after every two drops of PBS are washed.
Further, the preparation method in the sterilized PBST cleaning solution is to add 100ul TritonX100 to every 100ml PBS.
In the invention, the PBS is prepared according to the following proportion: taking 0.27g of monopotassium phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride, adding about 800mL of deionized water, fully stirring and dissolving, then adding concentrated hydrochloric acid to adjust the pH value to 7.4, finally fixing the volume to 1L, sterilizing at 121 ℃ for 20 minutes under high temperature and high pressure, and cooling for later use.
Preferably, in step S2, the amount of transfection mixture A added is at least 50ul, and the time for slow transfection on a shaker is 30min-1 h.
Preferably, in step S2, the whole culture solution C is used by changing the culture solution every 12 hours and changing the culture solution at least 3 times, so as to ensure that the total culture time of the whole culture solution C reaches 48 hours, and the whole culture solution C is washed at least once with PBS at room temperature every time the culture solution is changed.
Further, in the step S2, the transfection mixture A and the pre-culture mixture B were mixed to transfect Arm-Gal 4100 ng and UAS-GPI-GFP 100ng plasmid, and the total mass of the transfection plasmid was 200 ng.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention utilizes the digitonin (chemical cell membrane perforating agent) to assist in other protease inhibition and auxiliary culture reagents to directly perforate and penetrate cell membranes and nuclear membranes of tissues and organs of drosophila larvae, so that plasmids to be expressed are directly permeated into cell nuclei for expression without depending on cell division for transfection expression, thereby being suitable for cell lines and drosophila strains with proliferation and division obstacles, being capable of carrying out tissue and organ level transfection of drosophila larvae without being digested into single cells;
2. in the culture medium, energy metabolism substances of glutamine, pyruvic acid and sodium lactate are added to enhance the vitality of the tissue cells of drosophila larvae, which is beneficial to plasmid expression;
3. the method can be applied to the bacteria-bearing environment by adding multiple steps of intermediate liquid changing, so that the application range of the method is greatly improved, and the environmental cost is reduced;
4. the invention adds the step B of the pre-culture solution, so as to enhance the transfection infiltration effect of the plasmid;
5. the transfection efficiency is higher, according to immunofluorescence detection, the average transfection efficiency of an adult disc and a brain is improved by 1/3, and the average transfection efficiency of salivary glands is improved by 2/3;
6. the cost is low, the price of the whole set of reagent is about 5000 yuan, but the transfection of a sample exceeding 5000 can be realized.
Drawings
FIG. 1 is a diagram showing brain organs of Drosophila larvae 48 hours after transfection in the present invention;
FIG. 2 is an enlarged view of portion A of FIG. 1;
FIG. 3 shows the transfection effect of the salivary glands of Drosophila larvae.
Detailed Description
The foregoing aspects of the present invention are described in further detail by way of examples, but it should not be construed that the scope of the above-described subject matter is limited to the following examples, and that all the technologies implemented based on the above-described aspects of the present invention are within the scope of the present invention.
Examples
Preparation of PBS buffer: taking potassium dihydrogen phosphate (KH2PO 4): 0.27g, disodium hydrogen phosphate (Na2HPO 4): 1.42g, sodium chloride (NaCl): 8g of potassium chloride (KCl) and 0.2g of deionized water are added into about 800mL of deionized water and fully stirred for dissolution, then concentrated hydrochloric acid is added to adjust the pH value to 7.4, finally the volume is determined to be 1L, the mixture is sterilized at the high temperature and the high pressure of 121 ℃ for 20 minutes, and the mixture is cooled for standby.
Preparing a sterilized PBST cleaning solution: 100ul TritonX100 was added per 100ml of PBS buffer.
Preparing a transfection mixed liquor mother liquor: the main body is SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM (Sigma), 10ul glutamine (200mM), 2ul sodium lactate (60%), 10ul sodium pyruvate (100mM) are added into each 1ml of culture MEDIUM, and 1ug pepsin inhibitor (Pepstatin A), 10ug Aprotinin (Aprotinin) and 1ug Leupeptin (Leupeptin) are added into each ml of mother liquor. The culture medium is divided into 1ml and sodium pyruvate is stored in a freezing way at the temperature of-20 ℃ and is dissolved to room temperature before use. The glutamine and three protease inhibitor concentrated liquid are frozen and stored at-80 ℃, a small amount of glutamine and three protease inhibitor concentrated liquid is stored to-20 ℃ when the glutamine and three protease inhibitor concentrated liquid are temporarily used, the glutamine and three protease inhibitor concentrated liquid are used within one month, and the glutamine and three protease inhibitor concentrated liquid are overdue and discarded. The 60% sodium lactate storage solution is stored at 4 ℃.
Preparing a transfection mixed solution A: 100ul of transfection mixture mother liquor O +1ul of 5% digitonin (digitonin) + 200ng total amount of plasmid to be transfected, and digitonin is stored until-80 ℃ and-20 ℃ for 3 months. In this case, 100ng of each of the driver plasmids Arm-Gal4 and UAS-GPI-GFP was transfected.
Preparation of preculture solution B: 100ul SHIELDS AND SANG M3INSECT MEDIUM + 200ng total plasmid to be transfected.
Preparing a whole culture solution C: the main body is SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM, and each 900ul of the culture MEDIUM is added with 10ul of glutamine (200mM), 2ul of sodium lactate (60%), 10ul of sodium pyruvate (100mM), 10ul of penicillin streptomycin double-resistant concentrated stock solution (100X) and 100ul of fetal bovine serum.
An in-situ plasmid transfection method for drosophila larva tissue organs comprises the following steps:
1. the Drosophila three-instar larvae under common feeding conditions can be used in the experiment, and the accidental pollution probability can be reduced by feeding the Drosophila three-instar larvae under sterile or bacteria-reducing conditions.
2. About 10 fruit fly larvae of three years old are taken to be put into the sterilized PBST cleaning solution containing 0.1 percent TritonX-100, the surface is cleaned for 3 times, and no obvious dirt is ensured.
3. The sterilized PBS cleaning solution is used for dissecting drosophila larvae, so that intestinal tracts are not damaged as much as possible, and the content flows out to cause pollution. Intercepting the upper part of drosophila larvae, including eyes, wings, legs, balance bars, brain, central nervous system and salivary gland, and epidermis attached thereto. 10 drops of PBS (phosphate buffer solution) are dripped into the culture dish, each drop is about 60-80ul, and the culture dish is washed drop by drop, so that possible pollutants on the surfaces of tissues and organs and in gaps are washed as much as possible. The operation is carried out by sterilizing and cooling the alcohol lamp flame of the tweezers, and two drops are transferred once for sterilization.
4. The cleaned fruit fly tissue organ is transferred to 96-well culture dish, and each well can culture and transfect less than 5 larva tissue organs. Adding 50ul transfection mixture solution, and slowly transfecting on a shaker for 30min-1 h.
5. The transfection mixture A was washed rapidly with 100ul PBS for 2 times, several seconds each time.
6. And (4) completely sucking PBS, adding the preculture solution B, and culturing for 4-6 hours at 25 ℃.
7. The medium was changed to 200. mu.l of whole culture medium C, and then changed every 12 hours, and the medium was washed with room-temperature PBS to reduce the possibility of subsequent contamination. Placing the whole culture solution in a refrigerator at 4 deg.C, balancing to room temperature before use, and refrigerating. The culture time is more than 48 hours by changing the culture solution for 3 times.
8. Harvesting tissues and organs, fixing with formaldehyde, carrying out conventional immunofluorescence staining, and observing transfection effects.
Test examples
According to the transfection method of the above embodiment of the invention, the corresponding transfection effect monitoring is carried out on the air tube of the drosophila larva tissue.
As shown in FIG. 1, the two-knob (round bulge) cells showed widespread membrane-localized GFP expression 48 hours after transfection into the brain organ of Drosophila larvae. Two plasmids are transfected, one is an expression driving plasmid, Armadillo-Gal4, which has ubiquitous expression characteristics and can express a binary expression driving factor Gal4 after being accepted by almost all cells; another plasmid is UAS-GPI-GFP, the driver Gal4 bound to the regulatory element 5 × UAS is able to induce GPI-GFP expression in large amounts, GPI-GFP is a cell membrane-localized GFP with a phosphatidylinositol anchoring domain that exhibits a cell profile better than nuclear-or cytoplasmic-localized GFP and facilitates differentiation of cells that were successfully transfected.
FIG. 2 is an enlarged portion of the image shown in FIG. 1 within the white dashed box, where a large number of cells exhibiting a circular cell membrane localization and successful transfection can be observed.
FIGS. 3B and B' show the transfection effect of the salivary gland of Drosophila larvae, the transfection effect is stable, the successful transfection expression of the cells above 2/3 can be maintained, and the cells without green fluorescence cell membrane contour in the images are the cells which are not successfully transfected.
The transfection method can directly transfect the drosophila larva tissue organs, and can observe that the transfection and successful expression of exogenous genes of the drosophila adult disc, brain, salivary gland and other tissue organs can be realized within 48 hours. Because the technical scheme implements a rule of changing liquid within 12 hours and the tissue and organ culture of the drosophila melanogaster is 25 ℃, the proliferation speed of the microbial contamination is far from reaching the pollution degree, and the technology is suitable for a sterile operation environment and does not need to be operated among cells.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

Claims (6)

1. The in-situ plasmid transfection method for the tissues and organs of drosophila larvae is characterized by comprising the following steps:
s1 preparation of tissue and organ to be transfected by drosophila larvae: taking a plurality of fruit fly larvae as a group, repeatedly cleaning the fruit fly larvae in PBST for several times to remove dirt and pollutants outside the body surface, then dripping a sterilized PBS cleaning solution into a culture dish, dissecting the fruit fly larvae in the cleaning solution, and avoiding damaging intestinal tracts as much as possible to cause the pollution caused by the outflow of contents, and intercepting the upper sections of the fruit fly larvae, including eyes, wings, legs, balance bars, brains, central nervous systems, salivary glands and epidermis attached to the eyes, the wings, the legs, the balance bars, the brains and the central nervous systems;
s2 transfection: transferring the fruit fly tissue organ to a 96-hole culture dish, adding the transfection mixed solution A, and transfecting on a shaker at a slow speed; completely sucking the transfection mixed solution A, quickly cleaning the transfection mixed solution A by using PBS, adding the pre-culture solution B, and culturing for 4-6 hours at 25 ℃; replacing the pre-culture solution B with a full culture solution C for culture;
s3 transfection assay: harvesting the tissue and organ transfected by the step S2, fixing the tissue and organ with formaldehyde, and observing the transfection effect by adopting conventional immunofluorescence staining;
the preparation method of the transfection mixed solution A comprises the following steps: adding 1ul of 5% digitoxin concentrated stock solution and 200ng of plasmid to be transfected into 100ul of transfection mixed liquor mother liquor, wherein the main body of the transfection mixed liquor mother liquor is SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM, 10ul of 200mM glutamine, 2ul of 60% sodium lactate and 10ul of 100mM sodium pyruvate are added into 1ml of the culture MEDIUM, and 1ug of pepsin inhibitor, 10ug of aprotinin and 1ug of leupeptin are also added into 1ml of the transfection mixed liquor mother liquor;
the preparation method of the preculture solution B comprises the following steps: adding 200ng of plasmid to be transfected into 100ul SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM;
the preparation method of the whole culture solution C comprises the following steps: adding culture solution into SHIELDS AND SANG M3INSECT MEDIUM INSECT culture MEDIUM, wherein per 900ul culture MEDIUM is added with 10ul 200mM glutamine, 2ul 60% sodium lactate, 10ul 100mM sodium pyruvate, 10ul penicillin and streptomycin double-antibody concentrated stock solution and 100ul fetal calf serum, the concentration of penicillin is 10000U/ml, and the concentration of streptomycin is 10 mg/ml.
2. The in situ plasmid transfection method of drosophila larvae tissue organs according to claim 1, characterized in that: in step S1, the number of the larvae of the fruit flies in each group is not more than 5, the outer surfaces of the larvae of the fruit flies are washed repeatedly by PBST for 3 times, and the larvae of the fruit flies are dissected in a culture dish containing sterilized PBS.
3. The in situ plasmid transfection method of drosophila larvae tissue organs according to claim 1, characterized in that: in step S2, after dissection, the drosophila tissue organ was rapidly washed 2 times with PBS, and each washing process was as follows: after 10 dropwise washes with PBS wash and every two washes and transfers of tissue organs, the forceps were flame sterilized and cooled in air for use.
4. The in situ plasmid transfection method of drosophila larvae tissue organs according to claim 1, characterized in that: in step S2, the addition amount of the transfection mixture A is at least 50ul, and the slow transfection time on the shaker is 30min-1 h.
5. The in situ plasmid transfection method of drosophila larvae tissue organs according to claim 1, characterized in that: in step S2, the total culture solution C is used by changing the culture solution every 12 hours and changing the culture solution at least 3 times, so as to ensure that the total culture time of the total culture solution C reaches 48 hours, and the total culture solution C is washed at least once with PBS at room temperature every time the culture solution is changed.
6. The in situ plasmid transfection method of drosophila larvae tissue organs according to claim 1, characterized in that: in the step S2, 100ng of plasmid including Arm-Gal 4100 ng and UAS-GPI-GFP was transfected in the mixed transfection solution A and preculture solution B, and the total mass of the transfected plasmid was 200 ng.
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CN1365392A (en) * 1999-07-21 2002-08-21 犹太大学阿尔伯特爱因斯坦医学院 Drosophila recombination-associated protein and methods for use
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