Construction method of drosophila embryo permeabilization model
By treating Drosophila embryos with Citrasolv solution and bleaching agent solution, a highly permeable and viable Drosophila embryo permeation model was constructed, solving the toxicity problem of embryo removal caused by the wax layer in traditional methods, and enabling rapid and effective drug screening and toxicological evaluation.
Patent Information
- Application Number
- CN202511776026.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-28
- Publication Date
- 2026-02-17
AI Technical Summary
Existing technologies struggle to effectively remove the waxy layer to improve permeability while maintaining embryo viability and survival rate when constructing fruit fly embryo models. Traditional solvents such as heptane and octane are toxic and harmful to embryos.
Drosophila embryos were permeated with Citrasolv solution (mainly composed of limonene and surfactant). The waxy layer was removed by dilution to a ratio of 1:30 and treatment time of 2 minutes. The villous membrane layer was removed by bleaching solution, thus constructing a Drosophila embryo permeation model.
It achieves high permeability and high survival rate of Drosophila embryos, making them suitable for drug screening and toxicological evaluation. It enables rapid preparation of batches of permeable embryos, reduces damage to the embryos, and improves the efficiency and accuracy of drug screening.
Smart Images
Figure CN121538147A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing a Drosophila embryo permeation model. Background Technology
[0002] The fruit fly is one of the most thoroughly studied classic model organisms. Due to its high reproductive capacity, large number of offspring, rapid growth, short generation cycle, small size, ease of rearing and management, low chromosome number, diverse mutations, ease of observation and statistical analysis, clear genetic background, and rich phenotypic and developmental characteristics, it is a widely recognized model organism in the academic community. Numerous studies have confirmed that most of the mechanisms discovered in fruit flies are highly conserved in mammals.
[0003] The fruit fly embryo experiment is an in vivo experiment. Compared with general in vitro cultured cells and organs, fruit fly embryos develop more rapidly (at 25°C, fruit fly embryos develop into first-instar larvae in about 22 hours) and conform to a complete embryonic developmental state. Compared with mammalian embryos, fruit fly embryos are smaller, more numerous, easier to manipulate, and require less time to develop. Embryonic development at various stages can be observed under a microscope.
[0004] Drosophila embryos have become an important model for studying embryonic developmental biology. Histochemical staining, cytochemical labeling, the introduction of radiolabeled isotopes and other small molecules into the embryo will help improve our understanding of early embryogenesis. However, the Drosophila eggshell hinders the penetration of small molecules. From the outside in, the Drosophila eggshell consists of the outer chorionic membrane (300-500 nm), the endocrine layer (500-700 nm), the inner chorionic membrane (40-50 nm), the waxy layer (5 nm), and the vitelline membrane (300 nm thick). The main limitation on the entry of small molecules into the Drosophila embryo is the impermeability of the eggshell. The chorionic membrane can be effectively removed with 50% bleach without adversely affecting the embryo's survival rate. The waxy layer, also known as the "waterproof layer," lies beneath the inner chorionic membrane and surrounds the vitelline membrane, almost completely restricting embryo permeability. It is the final barrier for small solutes to enter the embryo, making the search for optimized methods to remove the waxy layer crucial. In 1973, Limbourg and Zalokar et al. used heptane and octane to dissolve the waxy layer, thus making de-egg-exposed embryos permeable. However, these solvents are toxic and reduce embryo viability. In 1992, Mazur et al. used rhodamine B dye to stain late-stage embryos treated with heptane, demonstrating that embryo viability is inversely proportional to permeability. Early embryos (stage 11 and earlier) are highly sensitive to this method, and it can potentially kill them directly. Currently, the construction of Drosophila embryo models has become increasingly sophisticated, but EPS (exfoliated polysaccharide) for removing the embryo's waxy layer still causes significant harm to the embryo. In 2010, Rand MD et al. used an embryo permeation solution composed of 90% D-limonene, 5% cocamide DEA, and 5% ethoxylated alcohol, demonstrating that solvents containing limonene are an ideal alternative to traditional heptane and octane for removing the waxy layer. Simultaneously, the permeability of the household product Citrasolv for ovariectomized embryos was evaluated, but only a 1:10 dilution was performed to determine a time range for embryo permeation. Under these conditions, while embryo permeability is increased, embryo survival rate is low. Therefore, this method can be further improved to enhance the penetration of small molecules into the embryo while minimizing damage to the embryo's viability.
[0005] With advancements in the synthesis of bioactive small molecules and the emergence of increasingly numerous synthetic compounds with unknown toxicity characteristics, analyzing the bioactivity of small molecules in Drosophila embryos is of great value for drug development and chemical synthesis. Currently, the Drosophila embryo model has evolved into an ideal model for neural development and drug screening, applied to studies of gene expression, mRNA localization, and immune tissue and cellular localization of proteins of interest. Therefore, exploring the process of Drosophila embryonic development is of great significance for revealing the cellular and molecular mechanisms of human embryonic development, elucidating the pathogenesis of various embryonic exposure-induced diseases, and in drug screening and toxicological evaluation. Summary of the Invention
[0006] The purpose of this invention is to provide a method for constructing a Drosophila embryo permeability model, which is simple and quick to operate, can ensure good embryo permeability, maintain embryo viability, facilitate the study of the effects of small molecule drugs on embryonic development, and also provides favorable conditions for studying the cellular and molecular mechanisms of embryonic development, toxicological evaluation of small molecule substances, and screening of relevant drugs in embryonic models.
[0007] To achieve the above objectives, the present invention provides a method for constructing a Drosophila embryo permeation model, comprising the following steps: Step 1, Collection and cleaning of fruit fly embryos: Collect fruit fly embryos 1-2 hours after birth, transfer the embryos into a nitex basket, and wash the embryos with a slow flow of warm tap water. Step 2, rinsing of fruit fly embryos: Use bleach solution to rinse the fruit fly embryos to remove the villi layer of the fruit fly embryos. After rinsing, wash the embryos with warm tap water at a slow flow rate. Step 3, Drosophila embryo permeation treatment: Drosophila embryos with the chorionic membrane layer removed are permeated in an embryo permeation solution EPS, and then washed in BPS to obtain a Drosophila embryo permeation model.
[0008] Furthermore, dyes were used to stain the Drosophila embryo infiltration model to verify the feasibility of Drosophila embryo infiltration.
[0009] Furthermore, in step 2, the bleaching solution is obtained by mixing an equal volume of sodium hypochlorite solution with a mass fraction of 5-6% and distilled water.
[0010] Furthermore, in step 2, the fruit fly embryos are rinsed with bleach solution for 1-5 minutes to remove the villi layer of the fruit fly embryos, preferably for 2 minutes.
[0011] Furthermore, in step 3, the embryo permeation solution EPS is Citrasolv, diluted with pure water at a ratio of 1:30, and the treatment time is 1-5 min.
[0012] To achieve the above objectives, the present invention also provides a Drosophila embryo permeation model, which is constructed by the above-described construction method.
[0013] This invention also provides the application of the above-mentioned Drosophila embryo permeation model in the study of cellular and molecular mechanisms, providing insights into cellular and molecular mechanisms related to embryonic development.
[0014] This invention also provides the application of the above-mentioned Drosophila embryo permeation model in the toxicological evaluation of small molecule substance screening, providing a basis for the toxicological evaluation of small molecule substance screening related to embryo development.
[0015] This invention also provides the application of the above-mentioned Drosophila embryo permeation model in screening for embryonic development toxic drugs.
[0016] Furthermore, in application, the test drug is added to the embryo culture medium of the Drosophila embryo permeation model, cultured to the required developmental stage, and the Drosophila embryo permeation model is subjected to immunofluorescence staining. The effect of the test drug on the embryo development process is observed using a laser confocal microscope, and relevant research and evaluation are carried out.
[0017] The construction method described in this invention can generate a large number of infiltrated embryos in a short time. Using non-infiltrated Drosophila embryos as a control, and with all other culture conditions identical, the embryos are stained with fluorescent dyes, and the dye penetration into the embryos is observed under a fluorescence microscope or a laser confocal microscope. Compared to the control group, if a large amount of dye enters the infiltrated embryos while having minimal impact on their viability, then this embryo manipulation method is considered a feasible method for embryo infiltration.
[0018] The method for drug toxicology evaluation using the Drosophila embryo permeation model is as follows: the test screening drug is added to the embryo culture medium, with an equal amount of blank solvent added as a control, and all other culture conditions are exactly the same. After culturing to the required developmental stage, the embryos are stained with immunofluorescence, and the effect of the test drug on the embryo development process is observed using a laser confocal microscope. If a distinguishable developmental defect occurs, the test screening drug is considered an embryo developmental toxic drug.
[0019] The advantages and positive effects of the method for constructing a Drosophila embryo permeation model described in this invention are as follows: 1. This invention establishes a Drosophila embryo permeation model that maintains embryo permeability while preserving embryo viability. Citrasolv, a concentrated household cleaner and degreaser, is mainly composed of limonene and surfactants, is miscible with water, and is less harmful to embryos compared to previous heptane and octane formulations.
[0020] 2. This invention can quickly and effectively perform large-scale embryo permeation treatment, allowing small molecule substances to enter the embryos, making the embryos a better model for small molecule drug screening.
[0021] 3. The embryos treated with permeation in this invention retain their morphological characteristics and exhibit good embryo viability. Developmental morphology diagrams of different embryonic stages can be obtained through staining or immunohistochemistry, allowing for clear observation of the entire process of Drosophila embryonic development. This lays the foundation for research on gene expression, mRNA localization, and immunohistocellular localization of proteins of interest during Drosophila embryonic development.
[0022] 4. In this invention, after EPS treatment, the Drosophila embryo permeation model maintained a high embryo viability, allowing for the assessment of various developmental endpoints after embryonic drug exposure. This procedure enables the direct and rapid preparation of batches of permeated embryos for toxicology studies and high-throughput screening of small molecules.
[0023] 5. When applied to drug toxicology evaluation, since the development time of fruit fly embryos is within 1 day, cell culture screening methods require at least 2 days, while traditional mammalian embryo development takes at least ten days and sometimes one or two years. Compared with traditional methods, this invention can perform preliminary screening of embryonic development-related drugs more quickly, and the screening results are more consistent with the actual situation.
[0024] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0025] Figure 1 This is an apparatus for collecting, rinsing, and permeating fruit fly embryos in an embodiment of the present invention. In the figure, 1 is an agar plate; 2 is yeast coated on the agar plate; 3 is a bottle; and 4 is a "nitex" basket. Figure 2 In this embodiment of the invention, Rhodamine 123 dye was used to stain the embryos to evaluate the effect of the embryo permeation solution on the permeation symptoms of Drosophila embryos. A is a microscopic image, where (a), (c), and (d) are images under a fluorescence microscope, and (b) is an image under an optical microscope. The scale bar is 500 μm. B is the hatching rate of Drosophila embryos treated with 1:30 EPS (+EPS) and untreated (-EPS). Figure 3 This invention is used in embodiments to evaluate the effect of embryo permeability solution treatment on embryo permeability and viability symptoms. Figure 4 In this embodiment of the invention, the use of BPF causes a sudden thinning or breakage of the longitudinal axis of the central nervous system, disrupting the integrity of the ventral nerve cord (VNC) structure and resulting in central nervous system developmental defects; A is DMSO treatment as a blank control; B is BPF as the experimental group. Figure 5The embodiments of this invention illustrate the effects of BPA, BPF, and BPS exposure on the growth and development of Drosophila, where A represents the effect of BPA on embryo hatching rate; B represents the effect of BPA on pupation rate; C represents the effect of BPA on emergence rate; D represents the effect of BPF on embryo hatching rate; E represents the effect of BPF on pupation rate; F represents the effect of BPF on emergence rate; G represents the effect of BPS on embryo hatching rate; H represents the effect of BPS on pupation rate; and I represents the effect of BPS on emergence rate. This indicates that P < 0.05. Detailed Implementation
[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0027] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0028] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.
[0029] This invention utilizes Citrasolv to remove the waxy layer of fruit fly embryos. Citrasolv, a common household cleaner, mainly contains limonene and surfactants, which effectively remove the waxy layer of fruit fly embryos with minimal damage to the embryos. A preferred dilution of the fruit fly embryo permeation solution is 1:20 to 1:40, with a treatment time of 2 minutes. A more preferred dilution is 1:30, with the embryos treated for 2 minutes.
[0030] Example 1: Construction of a Drosophila embryo permeation model 1. Collection and cleaning of fruit fly embryos: Fruit flies were cultured in an incubator at 25°C and 60% humidity. The fruit flies were transferred to a bottle with small holes around the edges, allowing them to lay their eggs on yeast cells on an agar plate. The fruit fly embryos were collected after 1-2 hours. Using a paintbrush, the embryos on the agar plate were transferred to a NITEX basket, and the embryos were washed with a slow-flowing stream of warm tap water.
[0031] 2. Washing of fruit fly embryos: Immerse the Nitex basket in a small beaker containing 20 mL of 50% bleach solution and rinse for 2 minutes, while simultaneously using a pipette to slowly rinse the embryos inside the Nitex basket with the bleach solution. After rinsing, wash the embryos with a slow flow of warm tap water.
[0032] 3. Drosophila embryo permeation treatment: Prepare EPS diluted 1:5 to 1:40 in a 50ml beaker and treat for 2 minutes, gently rinsing the embryos with EPS using a pipette during this time. Remove excess EPS from the bottom of the Nitex basket with a paper towel. Soak the basket in six beakers, each containing 10ml of PBS, to wash the embryos.
[0033] 4. Use dye treatment to assess embryo permeability: In a clear 1.5 ml microcentrifuge tube, add 500 μL of 1 mM Rh123 dye prepared with BPS. Gently transfer the embryo to the dye solution using a pen, and place the microcentrifuge tube on a vertical shaker and shake for 10 minutes. Remove the microcentrifuge tube and let it stand for 30 seconds to allow the embryo to settle. Open the microcentrifuge tube and aspirate the dye solution. Add BPS to the microcentrifuge tube, cap it, and invert it several times to wash the embryo. Allow the embryo to settle, aspirate the BPS, and repeat the BPS washing process three more times. Allow the embryo to settle and remove the BPS for use in embryo permeability identification and developmental analysis.
[0034] Figure 2 Image A shows embryos stained with Rhodamine 123 dye to evaluate the effect of embryo permeability solution on Drosophila embryo permeability symptoms. Embryos with stronger light intensity are those with higher permeability. Figure 2 In the middle A, 1:5 EPS, 1:10 EPS, the embryos with weak light are those with low permeability. Figure 1 In the control group (without EPS treatment), a dilution of 1:30 to 1:40 effectively improved embryo permeability while maintaining embryo viability. In the EPS (1:30) treatment group, the embryo hatching rate reached 85%. Figure 1 (B) Figure 3 To assess the effects of embryo permeability and viability after embryo treatment with the embryo permeability solution, a dilution ratio of 1:30 resulted in high levels of both embryo permeability and viability. Therefore, a dilution ratio of 1:30 was selected as the ideal EPS for drug toxicology evaluation.
[0035] Example 2: Rapid drug toxicology evaluation using a Drosophila embryo model. 1. Add the drug to be screened [e.g., 2 mM bisphenol F (BPF)] to the embryo culture medium, and use another portion of embryo culture medium with an equal amount of solvent dimethyl sulfoxide (DMSO) as a control.
[0036] 2. The embryos were developed to the observation endpoint (e.g., embryos at stage 14-15) in the culture medium for screening drugs, and then treated with immunofluorescence. The development of the central nervous system VNC was then detected by laser confocal microscopy.
[0037] 3. Comparison of VNC development in the embryonic central nervous system between the DMSO control group and the drug-treated group revealed that the VNC in the DMSO control group exhibited a normal "ladder-like" pattern with parallel longitudinal fasciculations and repeated anterior and posterior commissures within each segment, forming axonal framework. In contrast, the longitudinal axons in the BPF-treated group were thinner or broken. Figure 4 (As shown). Based on this, the following toxicological evaluation can be made of this drug: embryonic exposure to BPF disrupts the inherent morphology and structure of axonal guidance, thereby causing central nervous system developmental defects.
[0038] To assess the effects of embryonic exposure to BPA, BPF, and BPS on the growth and development of Drosophila, embryos treated with permeation therapy (1:30 dilution of EPS) were exposed to BPA, BPF, and BPS for 22 h. Hatching rate, pupation rate, and emergence rate of later larvae were analyzed. Results showed that compared to the control group, 0.5 mM exposure did not significantly affect embryo hatching rate, while 1 mM and 2 mM BPF exposure significantly reduced embryo hatching rate. Figure 5 Next, the effect of stopping BPF exposure during the larval stage on larval pupation was investigated. The results showed that BPF exposure during the embryonic stage did not have a significant effect on pupation rate. Figure 5 E). The eclosion rate was calculated based on the formation of pupae; compared with the control group, the eclosion rate of pupae showed no significant difference (E). Figure 5 These data indicate that high-dose BPF exposure reduced embryo hatching rate, but had no significant effect on pupation and emergence rates in Drosophila. Furthermore, the effects of embryonic BPA and BPS exposure on Drosophila growth and development were compared, and the results showed that neither BPA nor BPS had a significant effect on embryo hatching rate, pupation rate, or emergence rate. Figure 5 (Among AC and GI). This indicates that embryonic exposure to BPF has stronger developmental toxicity than BPA and BPS.
[0039] Therefore, the present invention adopts the above-mentioned method for constructing a Drosophila embryo permeation model, which is simple and quick to operate, can ensure good embryo permeability, maintain embryo viability, facilitate the study of the effects of small molecule drugs on embryonic development, and also provides favorable conditions for studying the cellular and molecular mechanisms of embryonic development, toxicological evaluation of small molecule substance screening, and screening of relevant drugs in embryonic models.
[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for constructing a fruit fly embryo permeabilized model, characterized by, The method comprises the following steps: Step 1, collection and cleaning of fruit fly embryos: collect fruit fly embryos 1-2 hours after birth, transfer the embryos into nitex baskets, and wash the embryos with slow-flowing warm tap water; Step 2, fruit fly embryo rinsing: use a bleaching solution to rinse the fruit fly embryos to remove the chorion layer of the fruit fly embryos, and after rinsing, wash the embryos with slow-flowing warm tap water; Step 3, fruit fly embryo permeabilization treatment: fruit fly embryos with the chorion layer removed are permeabilized in an embryo permeabilization solution EPS, and then washed in BPS to obtain a fruit fly embryo permeabilization model.
2. The method according to claim 1, wherein the method is characterized by: Use a dye to stain the fruit fly embryo permeabilization model to verify the feasibility of fruit fly embryo permeabilization.
3. The method according to claim 1, wherein the method is characterized by: In step 2, the bleaching solution is obtained by mixing equal volumes of a 5-6% sodium hypochlorite solution and distilled water.
4. The method of claim 1, wherein the method further comprises: (a) obtaining a Drosophila embryo; (b) permeabilizing the Drosophila embryo; and (c) fixing the permeabilized Drosophila embryo. In step 2, the bleaching solution is used to rinse the fruit fly embryos for 1-5 minutes to remove the chorion layer of the fruit fly embryos.
5. The method according to claim 1, wherein the method is characterized by: In step 3, the embryo permeabilization solution EPS is Citrasolv diluted with pure water at a ratio of 1:30, and the treatment time is 1-5 minutes.
6. A Drosophila embryo permeabilized model, characterized in that: The fruit fly embryo permeabilization model is constructed by the construction method of any one of claims 1-5.
7. Use of the Drosophila embryo permeabilized model according to claim 6 for the study of cellular and molecular mechanisms, characterized in that: To provide cell and molecular mechanisms related to embryonic development.
8. Use of the fruit fly embryo permeabilized model according to claim 6 for the toxicological evaluation of small molecule substances in a screening process, characterized in that: Toxicological evaluation of small molecule substance screening related to embryonic development.
9. The fruit fly embryo permeabilization model of claim 6 is used for screening embryonic development toxic drugs.
10. Use according to claim 7 or 8 or 9, characterized in that: In use, the fruit fly embryo permeabilization model is added with a drug to be tested in the embryo culture solution, cultured to the required development stage, immunofluorescently stained, observed under a laser confocal microscope to observe the influence of the drug to be tested on the embryonic development process, and related research and evaluation are carried out.