CN113930449B - Drosophila larva tissue organ in-situ plasmid transfection method - Google Patents

Drosophila larva tissue organ in-situ plasmid transfection method Download PDF

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CN113930449B
CN113930449B CN202111111905.2A CN202111111905A CN113930449B CN 113930449 B CN113930449 B CN 113930449B CN 202111111905 A CN202111111905 A CN 202111111905A CN 113930449 B CN113930449 B CN 113930449B
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transfection
solution
drosophila
tissue
organ
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CN113930449A (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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/103—Plasmid DNA for invertebrates
    • C12N2800/105—Plasmid DNA for invertebrates for insects

Abstract

The invention discloses an in-situ plasmid transfection method for tissues and organs of Drosophila larvae, and relates to the technical field of biology. The method comprises the following steps: s1, preparing tissues and organs to be transfected of Drosophila larvae; s2 transfection: transferring the Drosophila tissue and organ to a 96-hole culture dish, performing transfection on the mixed solution A after incubation and transfection in sequence, performing stable transfection on the pre-incubation culture solution B, and performing conventional culture on the transfected tissue and organ by using the whole culture solution C; s3 transfection detection: and (3) harvesting the tissue and organ transfected in the step (S2), fixing the tissue and organ by formaldehyde, and observing the transfection effect by adopting conventional immunofluorescence staining. The invention can directly transfect the fruit fly larva tissue organ, and the transfection and successful expression of exogenous genes can be observed in 48 hours, and the technical scheme is implemented by a 12-hour liquid change rule, and the fruit fly tissue organ is cultured at 25 ℃, so that the proliferation speed of microbial contamination can not reach the pollution degree, and the method is suitable for a bacterial operation environment.

Description

Drosophila larva tissue organ in-situ plasmid transfection method
Technical Field
The invention relates to the technical field of biology, in particular to an in-situ plasmid transfection method for tissues and organs of Drosophila larvae.
Background
Transfection (transfection) is the process of introducing exogenous DNA or RNA fragments actively or passively into cells in vitro or in vivo by corresponding biotechnological treatment, followed by protein expression or obtaining a new phenotype in the cells (e.g., RNAi). Conventional transfection techniques can be classified as transient transfection, in which exogenous DNA/RNA is not integrated into the host chromosome, and in which multiple copies of nucleic acid can be present simultaneously, and thus are high in expression levels and usually only maintain expression for several days, and stable transfection, in which exogenous DNA can be integrated into the host chromosome or possibly exist as episome.
Modern transfection methods are divided into: chemical, physical and viral transfection methods. Wherein the chemical method comprises the following steps: calcium phosphate method, DEAE-dextran method, liposome method, etc.; the physical method comprises the following steps: microinjection, electroporation, and gene gun; the chemical method is used for removing the calcium phosphate method, other types are required to use commercial transfection reagents, and the reagents have high cost of single transfection due to high price and large dosage, and in addition, the commercial transfection reagents can only be used for transfecting cell-level samples and are ineffective for tissues; while the physical laws require expensive instruments such as microinjection instruments, electrotransfection instruments, gene guns, and the like; the virus transfection method needs commercialized virus or self-packaging virus, has complicated operation flow, and the existing adenovirus transfection vector and the like are ineffective to drosophila cell tissues.
Meanwhile, in the prior art, the conventional transfection process generally only operates on cell-grade samples in a growing period, so that a high-standard sterile cell culture environment is required, and the conventional transfection process is almost ineffective on mutant cell strains with poor proliferation and division capacity and large tissue masses, and further increases the transfection cost.
Disclosure of Invention
The invention provides an in-situ plasmid transfection method for tissues and organs of Drosophila larvae, which 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 a Drosophila larva tissue organ comprises the following steps:
s1, preparing tissues and organs of Drosophila larvae: taking a plurality of Drosophila larvae as a group, repeatedly cleaning the Drosophila larvae in PBST for a plurality of times to remove dirt and pollutants outside the body surface, then dripping sterilizing PBS (phosphate buffer solution) cleaning solution into a culture dish, dissecting the Drosophila larvae in the cleaning solution, avoiding damaging intestinal tracts as much as possible, causing the outflow of contents to pollute, and intercepting the upper sections of the Drosophila larvae, including eyes, wings, legs, balance bars, brain, central nervous system, salivary glands and attached epidermis;
the three-instar larvae of Drosophila under common feeding conditions can be used in the experiment, and the probability of accidental pollution can be reduced by feeding under aseptic or bacteria-reducing conditions.
S2 transfection: transferring the tissues and organs of the drosophila melanogaster into a 96-hole culture dish, adding the transfection mixed solution A, and carrying out slow transfection on a shaking table; then sucking the transfection mixture A, using PBS for rapid cleaning, adding the preculture solution B, and culturing for 4-6 hours at 25 ℃; the preculture solution B is replaced by the whole culture solution C for culture; typically, less than 5 larval tissue organs per well can be cultured and transfected;
s3 transfection detection: harvesting the tissue and organ transfected in 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% digitalis glycoside concentrated stock solution and 200ng of plasmid to be transfected into 100ul of transfection mixed liquor mother solution, wherein the main body of the transfection mixed liquor mother solution is SHIELDS AND SANG M INSTCT MEDIUM INSECT culture MEDIUM, 10ul of 200mM glutamine, 2ul of 60% sodium lactate and 10ul of 100mM sodium pyruvate are added into each 1ml of culture MEDIUM, and in addition, 1ug of pepstatin, 10ug of aprotinin and 1ug of leupeptin are also added into each 1ml of transfection mixed liquor mother solution;
subpackaging insect culture medium into 1ml and storing sodium pyruvate in a concentrated way at-20deg.C for freezing, and dissolving to room temperature before use; the concentrated liquid of glutamine and three protease inhibitors is frozen and packaged and stored at-80 ℃, and is stored to-20 ℃ in a small amount when being temporarily used, and is used within one month and is discarded after expiration; preserving 60% sodium lactate storage solution at 4 ℃;
the digitalis glycoside concentrated stock solution is preserved to the condition of-80 ℃ and is effective at-20 ℃ for 3 months;
the preparation method of the preculture solution B comprises the following steps: 200ng of plasmid to be transfected was added to 100ul SHIELDS AND SANG M3INSECT MEDIUM insect medium;
the preparation method of the whole culture solution C comprises the following steps: to a main body of SHIELDS AND SANG M3.sup.3 INSTECT MEDIUM INSECT MEDIUM, 10ul 200mM glutamine, 2ul 60% sodium lactate, 10ul 100mM sodium pyruvate, 10ul 100X (preferably at a defined concentration, the concept of this ambiguity is not allowed in the patent) of penicillin and streptomycin double anti-concentrate stock and 100ul fetal bovine serum are added per 900ul MEDIUM.
The whole culture solution is placed in a refrigerator at 4 ℃ and is balanced to room temperature before use, and then is placed back for cold storage after use.
In the invention, in the step S1, the number of the Drosophila larvae in each group is not more than 5, the surfaces of the Drosophila larvae are repeatedly washed for 3 times by using PBST, and the Drosophila larvae are dissected in a culture dish containing sterilized PBS.
In the step S2, the dissected drosophila tissue and organ is quickly washed twice with PBS, and each washing process is as follows: after washing with PBS wash liquid for 10 times and washing and transferring the tissue and organ twice, the forceps were flame sterilized and cooled in air for use.
The specific operation can be as follows: and (3) dropwise adding 10 drops of PBS into the culture dish, wherein each drop is about 60-80ul, after dissection is completed, each tissue and organ of the drosophila larva is gradually cleaned, so that possible pollutants on the surface and in gaps of each tissue and organ of the drosophila larva are cleaned as much as possible, the drosophila larva is sterilized and cooled by using a tweezer alcohol burner flame for operation, and the tweezer needs to be sterilized once after every two drops of cleaning.
Further, the preparation method of the sterilized PBST cleaning solution is to add 100ul of TritonX100 to 100ml of 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 to 7.4, finally fixing the volume to 1L, sterilizing at the high temperature of 121 ℃ for 20 minutes, and cooling for later use.
Preferably, in the step S2, the amount of the transfection mixture A is at least 50ul, and the slow transfection time on a shaker is 30min-1h.
Preferably, in the step S2, the whole culture solution C is used for changing the solution once every 12 hours, and the solution is changed at least 3 times, so that the total culture time of the whole culture solution C is ensured to reach 48 hours, and the whole culture solution C is washed with PBS at room temperature at least once every time of changing the solution.
Further, in the step S2, the transfection solution A was mixed with the preculture solution B to transfect Arm-Gal4 100ng and UAS-GPI-GFP 100ng plasmids, and the total mass of the transfected plasmids was 200ng.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention uses chemical cell membrane perforation agent digitalis glycoside (digitonin) to assist in directly perforating and penetrating the cell membrane and nuclear membrane of the tissues and organs of the Drosophila larva by other protease inhibition and auxiliary culture reagents, so that the plasmid to be expressed directly permeates into the cell nucleus for expression without relying on cell division for transfection expression, thus being applicable to cell lines and Drosophila strains with proliferation and division disorder, and being capable of carrying out tissue organ level transfection of the Drosophila larva without being digested into single cells;
2. in the culture medium, energy metabolism substances of glutamine, pyruvic acid and sodium lactate are added, so that the activity of the tissue cells of the Drosophila larvae is enhanced, and plasmid expression is facilitated;
3. the added intermediate liquid change steps enable the method to be applied to a fungus environment, so that the application range of the application is greatly improved, and the environmental cost is reduced;
4. the pre-culture solution B is added, so that the plasmid transfection and infiltration effect can be enhanced;
5. the invention has higher transfection efficiency, the average transfection efficiency of the adult disc and the brain is improved by 1/3, and the average transfection efficiency of salivary glands is improved by 2/3 according to immunofluorescence detection;
6. the cost is low, the price of the whole set of reagent is about 5000 yuan, but the sample can be transfected by more than 5000 yuan.
Drawings
FIG. 1 shows the brain organs of Drosophila larvae after 48 hours of transfection in the present invention;
FIG. 2 is an enlarged schematic view of portion A of FIG. 1;
FIG. 3 shows the transfection effect of Drosophila larvae salivary glands.
Detailed Description
The above-described matters of the present invention will be further described in detail by way of examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and all techniques realized based on the above-described matters of the present invention are within the scope of the present invention.
Examples
PBS buffer was prepared: taking potassium dihydrogen phosphate (KH 2PO 4): 0.27g, disodium hydrogen phosphate (Na 2HPO 4): 1.42g, sodium chloride (NaCl): 8g of potassium chloride (KCl) 0.2g, adding about 800mL of deionized water, fully stirring and dissolving, then adding concentrated hydrochloric acid to adjust the pH to 7.4, finally fixing the volume to 1L, sterilizing at the high temperature of 121 ℃ for 20 minutes, and cooling for later use.
Preparing a sterilized PBST cleaning solution: 100ul TritonX100 was added to 100ml PBS buffer.
Preparing a transfection mixed solution mother solution: the main body was SHIELDS AND SANG M INSTECT MEDIUM INSECT MEDIUM (Sigma), 10ul of glutamine (200 mM), 2ul of sodium lactate (60%), 10ul of sodium pyruvate (100 mM) were added per 1ml of MEDIUM, and 1ug of Pepstatin (Pepstatin A), 10ug of Aprotinin (Aprotin) and 1ug of Leupeptin (Leupeptin) were added per ml of mother liquor. Subpackaging the culture medium into 1ml and concentrating sodium pyruvate for storage at-20deg.C, and dissolving to room temperature before use. The concentrated liquid of glutamine and three protease inhibitors is frozen and packaged and stored at-80 ℃, and is stored to-20 ℃ in a small amount when being temporarily used, and is used within one month and is discarded after expiration. 60% sodium lactate stock solution was stored at 4 ℃.
Preparing transfection mixed solution A:100ul of transfection mixture stock O+1ul 5% digitonin stock (digitonin) +200 ng of total amount of plasmid to be transfected, digitonin stock is stored at-80℃for 3 months to be effective at-20 ℃. In this case, the transfection-driven plasmids Arm-Gal4 and UAS-GPI-GFP were each 100ng.
Preparation of preculture solution B:100ul SHIELDS AND SANG M3INSECT MEDIUM insect medium+200 ng total plasmid to be transfected.
Preparing a whole culture solution C: the main body is SHIELDS AND SANG M INSTECT MEDIUM INSECT culture MEDIUM, and 10ul glutamine (200 mM), 2ul sodium lactate (60%), 10ul sodium pyruvate (100 mM), 10ul penicillin streptomycin double anti-concentrated stock solution (100X) and 100ul fetal bovine serum are added into each 900ul of culture MEDIUM.
An in-situ plasmid transfection method for a Drosophila larva tissue organ comprises the following steps:
1. the three-instar larvae of Drosophila under common feeding conditions can be used in the experiment, and the probability of accidental pollution can be reduced by feeding under aseptic or bacteria-reducing conditions.
2. About 10 three-year-old Drosophila larvae are taken into a sterilized PBST cleaning solution containing 0.1% TritonX-100, and the surfaces are cleaned for 3 times, so that no obvious dirt is ensured.
3. And the sterilizing PBS cleaning solution is used for dissecting the Drosophila larvae, so that intestinal tracts are prevented from being damaged as much as possible, and the outflow of the content causes pollution. The upper segment of Drosophila larvae, including the tissues and organs of eyes, wings, legs, balance bars, brain and central nervous system, salivary glands, and the epidermis to which they are attached, is intercepted. 10 drops of PBS are dripped into the culture dish, each drop is about 60-80ul, the washing is carried out drop by drop, and possible pollutants on the surfaces and gaps of the tissues and organs are washed as much as possible. The alcohol lamp flame disinfection and cooling operation is carried out on tweezers, and the alcohol lamp is used after being disinfected once every two drops are transferred.
4. The cleaned Drosophila tissue organs were transferred to 96-well petri dishes, and less than 5 larva tissue organs were cultured and transfected per well. 50ul of the transfection mixture A is added, and the mixture is slowly transfected on a shaking table for 30min-1h.
5. The transfection mixture A was blotted and rapidly washed 2 times with 100ul of PBS for several seconds.
6. The PBS is sucked and added into the preculture solution B for culturing for 4 to 6 hours at 25 ℃.
7. The solution was changed to 200. Mu.l of the whole culture solution C, and then the solution was changed every 12 hours, and the solution was washed with PBS at room temperature to reduce the possibility of subsequent contamination. The whole culture solution is placed in a refrigerator at 4 ℃ and is balanced to room temperature before use, and then is placed back for cold storage after use. The liquid is exchanged for 3 times, so that the culture time reaches more than 48 hours.
8. Harvesting tissue and organ, fixing by formaldehyde, staining by conventional immunofluorescence, and observing transfection effect.
Test examples
According to the transfection method of the embodiment of the invention, the corresponding transfection effect monitoring is carried out on the tissue trachea of the Drosophila larvae.
As shown in FIG. 1, the Drosophila larval brain organs 48 hours after transfection, the cells of the two-optic bulb part (circular enlargement) showed a general membrane-localized GFP expression. Two plasmids, one of which is an expression driving plasmid, armadillio-Gal 4, which has ubiquitous expression characteristics and can express binary expression driving factor Gal4 after almost all cells are accepted; another plasmid is UAS-GPI-GFP, and binding of driver Gal4 to regulatory element 5 XUAS induces GPI-GFP expression in large amounts, GPI-GFP being a cell membrane-localized GFP with phosphatidylinositol anchoring domains, displaying more cell contours than nuclear-localized or cytoplasmic localized GFP and facilitating differentiation of transfected cells.
FIG. 2 is an enlarged portion of the image shown within the box of the white dashed line in FIG. 1, showing that a large number of cells exhibiting circular membrane localized transfection success were observed.
FIGS. 3B and B' show transfection effect of Drosophila larvae salivary glands, the transfection effect is stable, and cells with no outline of green fluorescent cell membrane in the image are untransfected functional cells, which can keep over 2/3 of cells successfully transfected and expressed.
The transfection method can directly transfect tissues and organs of the Drosophila larvae, and can observe that the tissues and organs of the adult disc, brain, salivary glands and the like of the Drosophila achieve transfection and successful expression of exogenous genes in 48 hours. Because the technical proposal is implemented for 12 hours with regular liquid exchange, and the fruit fly tissue and organ culture is 25 ℃, the proliferation speed of microbial contamination can not reach the pollution degree, so the technology is suitable for the bacterial operation environment without the operation among cells.
The above 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 color changes made in the main design concept and spirit of the present invention are still consistent with the present invention, and all the technical problems to be solved are included in the scope of the present invention.

Claims (6)

1. The in-situ plasmid transfection method for the tissues and organs of the Drosophila larvae is characterized by comprising the following steps of:
s1, preparing tissues and organs to be transfected by Drosophila larvae: taking a plurality of Drosophila larvae as a group, repeatedly cleaning the Drosophila larvae in PBST for a plurality of times to remove dirt and pollutants outside the body surface, then dripping sterilizing PBS (phosphate buffer solution) cleaning solution into a culture dish, dissecting the Drosophila larvae in the cleaning solution, avoiding damaging intestinal tracts as much as possible, causing the outflow of contents to pollute, and intercepting the upper sections of the Drosophila larvae, including eyes, wings, legs, balance bars, brain, central nervous system, salivary glands and attached epidermis;
s2 transfection: transferring the tissues and organs of the drosophila melanogaster into a 96-hole culture dish, adding the transfection mixed solution A, and carrying out slow transfection on a shaking table; then sucking the transfection mixture A, using PBS for rapid cleaning, adding the preculture solution B, and culturing for 4-6 hours at 25 ℃; the preculture solution B is replaced by the whole culture solution C for culture;
s3 transfection detection: harvesting the tissue and organ transfected in 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% digitalis glycoside concentrated stock solution and 200ng of plasmid to be transfected into 100ul of transfection mixed liquor mother solution, wherein the main body of the transfection mixed liquor mother solution is SHIELDS AND SANG M INSTCT MEDIUM INSECT culture MEDIUM, 10ul of 200mM glutamine, 2ul of 60% sodium lactate and 10ul of 100mM sodium pyruvate are added into each 1ml of culture MEDIUM, and in addition, 1ug of pepstatin, 10ug of aprotinin and 1ug of leupeptin are also added into each 1ml of transfection mixed liquor mother solution;
the preparation method of the preculture solution B comprises the following steps: 200ng of plasmid to be transfected was added to 100ul SHIELDS AND SANG M3INSECT MEDIUM insect medium;
the preparation method of the whole culture solution C comprises the following steps: to a main body of SHIELDS AND SANG M3.sup.3 INSTECT MEDIUM INSECT MEDIUM, 10ul 200mM glutamine, 2ul 60% sodium lactate, 10ul 100mM sodium pyruvate, 10ul penicillin and streptomycin double anti concentrate stock solution and 100ul fetal bovine serum were added per 900ul MEDIUM, wherein the penicillin concentration was 10000U/ml and the streptomycin concentration was 10mg/ml.
2. The method for in situ plasmid transfection of Drosophila larva tissue organs according to claim 1, wherein: in step S1, the number of Drosophila larvae in each group is not more than 5, the surfaces of the Drosophila larvae are repeatedly washed 3 times by PBST, and dissected in a culture dish containing sterilized PBS.
3. The method for in situ plasmid transfection of Drosophila larva tissue organs according to claim 1, wherein: in the step S2, the dissected drosophila tissue and organ is quickly washed 2 times with PBS, and each washing process is as follows: after washing with PBS wash liquid for 10 times and washing and transferring the tissue and organ twice, the forceps were flame sterilized and cooled in air for use.
4. The method for in situ plasmid transfection of Drosophila larva tissue organs according to claim 1, wherein: in the step S2, the addition amount of the transfection mixture A is at least 50ul, and the slow transfection time on a shaking table is 30min-1h.
5. The method for in situ plasmid transfection of Drosophila larva tissue organs according to claim 1, wherein: in the step S2, the whole culture solution C is used for changing the solution once every 12 hours, and the solution is changed at least 3 times, so that the total culture time of the whole culture solution C is ensured to reach 48 hours, and the whole culture solution C is washed at least once by using room temperature PBS every time of the solution change.
6. The method for in situ plasmid transfection of Drosophila larva tissue organs according to claim 1, wherein: in the step S2, the transfection solution A and the preculture solution B are mixed to transfect Arm-Gal4 100ng and UAS-GPI-GFP 100ng plasmids, and the total mass of the transfected plasmids is 200ng.
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