A culture method of double fluorescent labeled caenorhabditis elegans for monitoring mitochondrial homeostasis

Abstract

The application belongs to the field of biotechnology and genetic engineering, and discloses a culture method of double-fluorescently labeled Caenorhabditis elegans for monitoring mitochondrial homeostasis, which comprises the following steps: culturing Caenorhabditis elegans ATU3301 strain to L4 larva stage, and selecting male as the father; culturing Caenorhabditis elegans MQD753 strain to L4 larva stage, and selecting hermaphrodite as the mother; placing them on a culture plate in a certain proportion, mating, and obtaining offspring; removing the parents and continuing to culture the offspring to L2 larva stage; screening Caenorhabditis elegans through a microscope to obtain target Caenorhabditis elegans; without an external staining agent, the fluorescent protein is stably integrated into the genome and specifically located in mitochondria, the signal is stable, the location is accurate, and the repeatability is high, which provides a new tool with physiological relevance, operational convenience and cost advantage for the research on mitochondrial homeostasis mechanism and the development of related disease drugs, and has great potential application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biotechnology, and specifically to a method for culturing Caenorhabditis elegans with dual fluorescent labels for mitochondrial homeostasis monitoring. Background Technology

[0002] Mitochondria, as the cell's energy factories and signaling hubs, play a crucial role in maintaining cellular homeostasis. Imbalances in mitochondrial homeostasis, such as disordered mitochondrial calcium signaling, excessive activation by oxidative stress, disruption of kinetic equilibrium, and impaired autophagy clearance, directly lead to the collapse of mitochondrial energy metabolism and the accumulation of mtDNA damage, subsequently triggering or exacerbating a range of diseases, including neurodegenerative diseases, metabolic diseases, cardiovascular diseases, and age-related diseases. However, current technologies for detecting mitochondrial homeostasis have limitations. Most methods focus on a single indicator of mitochondrial function and are static endpoints, failing to capture the dynamic changes in multiple synergistic pathological or physiological processes. While mammalian models can partially simulate the in vivo pathological microenvironment, they suffer from ethical restrictions, long experimental cycles, and high costs. In vitro cell models struggle to simulate the real-world mitochondrial homeostasis regulatory mechanisms and pathophysiological processes, making them unsuitable for screening multi-component, multi-target formulations in traditional Chinese medicine. Therefore, developing a real-time, convenient model for monitoring mitochondrial homeostasis is of significant value for basic research and drug development.

[0003] *C. elegans*, due to its transparent body structure, fixed cell lineage, and convenient genetic manipulation capabilities, along with its short lifespan and significantly lower cost compared to mammalian models, is an important model organism in life science research. Mitochondrial calcium homeostasis and mitochondrial oxidative stress are core functional indicators reflecting mitochondrial homeostasis, and their synergistic detection can rapidly determine mitochondrial imbalance. Currently, several *C. elegans* strains are available for monitoring specific mitochondrial functions. For example, the MQD753 strain carries the mitochondrial localization sequence of SDHB-1, while cpYFP, ​​a circularly arranged yellow fluorescent protein, is a superoxide sensor that can be used to detect mitochondrial superoxide. The ATU3301 strain carries mitochondrial LAR-GECO, a gene-encoded red fluorescent calcium ion indicator located in mitochondria, which can be used to detect mitochondrial calcium ions. However, in complex physiological and pathological processes, mitochondrial oxidative stress and calcium signaling often interact and change synergistically. Traditional methods require co-culturing two single-labeled strains and then observing them separately. This not only makes it impossible to simultaneously track the dynamic correlation between calcium signaling and oxidative stress in the same nematode individual, but also makes it difficult to eliminate the interference of individual differences on the test results. Staining with probes and dyes is also problematic due to issues such as the toxicity and inconsistent penetration of the staining agents and signal quenching, which severely limit the in-depth analysis of the mitochondrial homeostasis regulation mechanism and cannot meet the requirements of high-throughput drug screening.

[0004] To address the aforementioned technical problems, this invention provides a method for culturing *C. elegans* with dual fluorescent labeling for mitochondrial homeostasis monitoring. Using this method, a novel dual fluorescent labeling *C. elegans* strain, GCM1101, was cultured. This strain not only stably integrates a mitochondrial-localized calcium signaling indicator and a mitochondrial-localized oxidative stress sensor into the same nematode, enabling simultaneous and dynamic tracking of changes in mitochondrial calcium concentration and superoxide levels, and clearly observing their correlation, but also significantly outperforms traditional staining methods in terms of signal stability, experimental convenience, and dynamic monitoring capabilities. It is a valuable tool for mitochondrial function research. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for culturing *C. elegans* with dual fluorescent labels for mitochondrial homeostasis monitoring, characterized by comprising the following steps: (1) Cultivate the ATU3301 strain of Caenorhabditis elegans to the L4 larval stage and select males as the male parent; (2) Cultivate the Caenorhabditis elegans MQD753 strain to the L4 larval stage, and select hermaphroditic individuals as the female parent; (3) Place them on a culture plate in proportion, mate them, and obtain offspring; (4) Remove the parent and continue to raise the offspring to the L2 larval stage; (5) The target Caenorhabditis elegans was obtained by screening Caenorhabditis elegans under a microscope.

[0006] Preferably, the ratio described in step (3) is 1:1 to 1:3.

[0007] Preferably, the culture plate in step (3) is an NGM culture plate.

[0008] Preferably, the microscope screening in step (5) includes stereomicroscope screening and fluorescence microscope screening.

[0009] Preferably, the stereomicroscopy screening of nematodes with a rolling phenotype and the fluorescence microscopy screening, based on the stereomicroscopy screening, screen for nematodes whose pharyngeal muscles simultaneously emit cpYFP green fluorescence and RFP red fluorescence, and whose body walls have LAR-GECO red fluorescence.

[0010] Preferably, the target Caenorhabditis elegans genome described in step (5) is stably integrated with the hqIs180 [sdhb-1p::mtLS::cpYFP+rol-6(su1006)] genetic element from the MQD753 strain and the aceIs1 [myo-3p::mitochondrial LAR-GECO + myo-2p::RFP] genetic element from the ATU3301 strain.

[0011] Compared to existing technologies, the advantages of this invention are as follows: This invention provides a method for culturing *C. elegans* with dual fluorescent labeling for mitochondrial homeostasis monitoring through a hybridization strategy. The strain can simultaneously carry a mitochondrial superoxide sensor (cpYFP) and a mitochondrial calcium ion indicator (LAR-GECO) integrated into a single in vivo model, enabling simultaneous real-time monitoring of two key mitochondrial homeostasis indicators. After rigorous hybridization screening and multi-generational genetic stability verification, both the genotype and phenotype are stably inherited, ensuring the reliability, reproducibility, and consistency of experimental results. This strain requires no exogenous staining agents, avoiding problems such as dye toxicity, uneven penetration, rapid quenching, and strong background interference. The fluorescent protein is stably integrated into the genome and specifically located in mitochondria, providing stable signals, precise localization, and high reproducibility. It is highly suitable for constructing high-throughput drug screening platforms. This strain can be used to discover drugs or compounds that can regulate mitochondrial function and alleviate oxidative stress and calcium overload, providing a novel tool with physiological relevance, ease of operation, and cost advantages for mitochondrial homeostasis mechanism research and related disease drug development, possessing enormous potential application value.

[0012] Leveraging the transparent nature of nematodes and combining it with confocal microscopy, the spatial distribution of fluorescence signals can be directly observed, allowing for the exploration of different mitochondrial states in different locations. Nematode culture is low-cost and rapidly reproduces, enabling large-scale sample screening. Furthermore, RNAi interference, gene knockout, and other techniques can be used in conjunction with dual fluorescence signals for precise localization of key genes or pathways regulating mitochondrial calcium-oxidative stress balance, providing a more accurate tool for disease mechanism research. The construction of this strain fills the technological gap in existing models that cannot simultaneously monitor core mitochondrial functional indicators, providing an efficient and convenient new research tool for mitochondrial homeostasis research and the development of targeted drugs for related diseases. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0014] Figure 1 A schematic diagram illustrating the process of constructing hybrid strains of Caenorhabditis elegans.

[0015] Figure 2 Bright-field and fluorescence phenotypic images of hybrid nematodes MQD753, ATU3301, and GCM1101.

[0016] Note: N2: Wild type (non-fluorescent) is the blank control group; ATU3301: Pharyngeal pump expression of LAR-GECO is shown in red; MQD753: Somatic wall pharyngeal pump expression of cpYFP; The GCM1101 hybrid line has both pharyngeal pump expression of LAR-GECO and somatic wall pharyngeal pump expression of cpYFP. TL indicates bright field; Merge indicates colocalization (yellow). Scale bar = 100 μm.

[0017] Figure 3 Schematic diagram of PCR sequencing of hybrid nematodes MQD753, ATU3301, and GCM1101.

[0018] Note: (A) PCR amplification results of the hqIs180 gene in the MQD753 strain. (B) PCR amplification results of the ccIs4251 gene in the ATU3301 strain. (C) PCR amplification results of the aceIs1 gene in the ATU3301 strain.

[0019] Figure 4 The effects of hybrid nematodes MQD753, ATU3301, and GCM1101 on body length and width.

[0020] Figure 5 Effects of hybrid nematodes MQD753, ATU3301, and GCM1101 on head twitching frequency, pharyngeal pump, and total number of eggs laid.

[0021] Figure 6 The effects of hybrid nematodes MQD753, ATU3301, and GCM1101 on lifespan.

[0022] Note: Figures 4 to 6 Each experiment (n = 15) was conducted three independent trials. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference.

[0023] Figure 7 ROS and mitochondrial calcium fluorescence images of N2 and GCM1101 nematodes.

[0024] Note: N2 staining group (exogenous MitoSOX and Rhod-2 / AM staining); GCM1101 group (with built-in cpYFP green superoxide probe + LAR-GECO red calcium probe). TL bright field; GFP labeled mitochondrial superoxide; Cy3 labeled mitochondrial calcium ions; Merge for dual signal colocalization (yellow). Scale bar = 100 μm. The N2 exogenous staining group showed significant background interference, while the GCM1101 built-in fluorescent group showed clear signals and accurate localization. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] In the following examples, the directional terms left, right, up, down, and center are all literal directional terms.

[0027] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature within this field or in accordance with the product instructions. Reagents, consumables, or instruments whose manufacturers are not specified are all conventional products that can be purchased from the market.

[0028] Example 1: Construction and Screening of the GCM1101 Line for Specificity 1. Materials 1.1 Nematode strains N2 is C. elegans Wild isolates; ATU3301(ccIs4251 [myo-3p::GFP::LacZ::NLS+ myo-3p::mitochondrial GFP+dpy-20(+)]I.aceIs1[myo-3p::mitochondrial LAR-GECO+myo-2p::RFP]); MQD753(hqIs180[sdhb-1p::mtLS::cpYFP+rol-6(su1006)]); uracil-leaking mutant E. coli OP50 were all purchased from Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, USA).

[0029] 1.2 Medicines and Reagents Levamisole hydrochloride: Product code HY-13666, purchased from MedChemexpress Biotechnology Company, USA, concentration 0.5%.

[0030] 1.3 Instruments and Equipment Incubator (LRH-150, Shanghai Yiheng Scientific Instruments Co., Ltd., China); Stereo microscope (SZ660, Chongqing Aote Optical Instruments Co., Ltd., China); Inverted fluorescence microscope (A xioVert5, Carl Zeiss AG, Germany).

[0031] 2. Methods 2.1 Culture and Synchronization of Caenorhabditis elegans Using a pick, streak OP50 bacterial suspension onto LB agar plates and incubate at 37°C for 12-14 hours. Pick single colonies of OP50 and transfer them to LB liquid medium, incubate at 37°C with shaking for 12-14 hours, then store at 4°C for later use. After solidification, evenly spread the OP50 bacterial suspension onto NGM medium and incubate at 37°C for 12-14 hours, then store at 15°C for later use.

[0032] Remove the frozen nematodes from the freezer, thaw them rapidly in a 37°C water bath, pour them into a new centrifuge tube, rinse with M9 solution to remove the cryoprotectant, and let stand for 1 hour. Take the nematode pellet from the bottom of the centrifuge tube and add it to an NGM medium inoculated with OP50 Escherichia coli bacteria. Incubate at 16°C for later use.

[0033] Nematode synchronization was achieved using the sodium hypochlorite lysis method. Nematodes cultured on NGM medium were collected using M9 buffer, lysis buffer was added, and the mixture was vortexed for 5-7 min until complete lysis of the adults. The mixture was then centrifuged at 5000 rpm for 1 min, the supernatant was discarded, and the eggs were washed twice with M9 buffer to obtain nematode eggs. These eggs were then incubated at a suitable temperature according to the nematode culture requirements to obtain synchronized nematodes.

[0034] 2.2 Hybridization Experiment Nematodes are generally hermaphroditic, with a male ratio of 0.05%. However, hybridization between males and hermaphrodites can produce 950-1050 offspring, while hermaphrodites only produce 250-300. Hermaphrodites must be selected from L4 stage males (with a clear semi-circular bright spot on the abdomen) of suitable age. The MQD753 and ATU3301 strains were synchronized using the sodium hypochlorite lysis method to obtain L0 stage nematodes. After culturing in M9 medium for 12-24 hours until reaching the L1 stage, these nematodes were inoculated onto NGM plates containing E. coli OP50 and cultured in a constant temperature incubator until reaching the L4 larval stage. Healthy and vigorous individuals were selected for subsequent hybridization experiments. Male nematodes of the L4 stage ATU3301 strain and hermaphroditic nematodes of the MQD753 strain, or hermaphroditic nematodes of the ATU3301 strain and male nematodes of the MQD753 strain, were transferred to the same small NGM medium at a specific ratio (hermaphroditic to male nematodes should be controlled between 1:1 and 3:1) to optimize hybridization efficiency, facilitate operation, and reduce interference. Six culture dishes were replicated, and the nematodes were cultured at 20°C for 3-4 days. The hybridization success rate was calculated. After oviposition, the parent nematodes were removed, and the offspring eggs were allowed to continue developing at 20°C to the L2 larval stage (e.g., ...). Figure 1 (As shown).

[0035] 2.3 Offspring Screening L2 stage nematodes were first observed phenotyped, and after screening for the rolling phenotype, they were observed using an inverted fluorescence microscope. Among them, the O phenotype in the MQD753 strain... -2 The specific fluorescent probe cpYFP is excited at 488 nm and emitted at 515 nm. In the ATU3301 strain, mitochondrial LAR-GECO is excited at 557 nm and emitted at 584 nm; RFP is excited at 532 nm and emitted at 588 nm. After anesthesia with 0.5% levamisole hydrochloride, fluorescence was observed under an inverted fluorescence microscope. The core screening criterion was that both the yellow-green fluorescence signal of cpYFP and the red fluorescence signal of RFP must be observed simultaneously in the pharyngeal region of the same nematode, and the red fluorescence of LAR-GECO must be observed in the body wall region. Individuals with only a single fluorescent signal were excluded.

[0036] 3. Results The experimental results showed that more eggs were produced in the three culture dishes, with a hybridization success rate of approximately 50%. The nematode phenotype should be considered when performing hybridization. The parental nematodes were removed from the plates, leaving the plates containing progeny eggs to continue culturing until they reached the L2 stage for photographing. It was found that some nematodes exhibited the rolling phenotype of the MQD753 strain, and their pharyngeal pumps showed green and red fluorescence, while their body walls showed red fluorescence. Figure 2 The results showed that the hybridization was successful. Thirty offspring were randomly selected to calculate the heritability. The results showed that the heritability of culture dishes 1-3 was 60%, 60%, and 63.3%, respectively (Table 1), indicating that the hybrid offspring had a high success rate of inheritance.

[0037] Table 1. Cross-linked heritability table

[0038] Example 2: Genotyping Verification and Stability Establishment of the GCM1101 Line 1. Method 1.1 PCR reaction system and procedure The hybrid nematodes selected from the offspring were subjected to PCR verification and sequencing to extract genomic DNA from N2, ATU3301, MQD753, and GCM1101. The total volume of the PCR reaction system was 10 μL. 0.5 μL of nematode lysate was used as a template, and 5 μL of 2×Rapid Taq Master Mix was added. Then, 0.05 μL of 100 μM upstream primer and 0.05 μL of the same concentration downstream primer were added, and finally, 4.4 μL of enzyme-free water was added to bring the total volume to 10 μL. The PCR reaction program was set as follows: First, the reaction system was pre-denatured at 95℃ for 3 min; then, the cycling phase was started, and a total of 30 cycles were run. Each cycle included denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and extension at 72℃ (the extension time was calculated according to the product length, with 6 seconds corresponding to each kb fragment); after the cycling, a final extension was performed at 72℃ for 5 min; finally, the system was maintained at 18℃ to complete the entire program. Primer design: Specific primers were designed for the hqIs180 gene of MQD753 strain, the ccIs4251 gene and aceIs1 gene of ATU3301 strain (Table 2). The PCR products were detected by agarose gel electrophoresis and then verified by Sanger sequencing.

[0039] Table 2 Primer Sequences

[0040] 1.2 Validation of genetic stability The successfully verified hybrid nematodes were continuously passaged. Once they reached the L2 stage, their phenotype was observed, and 30 nematodes were randomly selected to calculate their heritability. This process was repeated three times. The heritability of the three generations was calculated.

[0041] 2. Results In the PCR detection, the lanes from left to right are: hybrid nematode strains 1-8, MQD753, ATU3301, N2, and Mark. The PCR amplification results of the hybrid nematodes and MQD753 were as expected, successfully obtaining the 848bp target fragment hqIs180; the target fragment was not detected in ATU3301 and N2, indicating that the hybrid nematodes successfully integrated the mitochondrial oxidative stress-related fluorescent gene fragment hqIs180. Figure 3 A). The target fragment ccIs4251 (1033 bp) was not found in any lane of the hybrid nematode strain, suggesting that this fragment failed to integrate into the hybrid nematode genome during hybridization due to genetic recombination, fragment loss, or other reasons. Figure 3B). The PCR amplification results of the hybrid nematodes and ATU3301 were as expected, and the target fragment aceIs1 of 1338 bp was successfully obtained; the target fragment was not detected in MQD753 and N2, indicating that the hybrid nematodes successfully acclimated the fluorescent gene for mitochondrial calcium. Figure 3 C). Therefore, the simultaneous presence of fluorescent markers for mitochondrial oxidative stress and mitochondrial calcium in the same nematode indicates successful hybridization. Genetic stability verification results showed that after at least three consecutive generations, the core phenotype and genotype heritability remained at 100% (Table 3), demonstrating good genetic stability. Therefore, it was established and named the GCM1101 strain.

[0042] Table 3. Heritability of phenotypes in hybrid nematodes of different generations

[0043] Example 3: Phenotypic and physiological verification of the GCM1101 strain To confirm that the hybridization process did not cause unintended toxicity to the nematodes and to ensure that the GCM1101 strain was a healthy experimental model, we measured its key phenotypes and physiological indicators.

[0044] 1. Medicines and reagents FUDR: Product No. F809658, purchased from Shanghai Yuanye Biotechnology Co., Ltd., working solution concentration is 1mg / mL, prepared and used immediately.

[0045] 2. Methods The synchronized N2, GCM1101, MQD753, and ATU3301 lines were cultured under the same conditions.

[0046] Developmental and morphological indicators: In the late L4 stage, images were taken under a stereofluorescence microscope after anesthesia, and the body length and width of the adults were measured using ImageJ software, with 30 adults per group.

[0047] Motor function indicators: In the late L4 stage, 60 μL of M9 buffer was added to the surface of the culture medium in the test tray. Nematodes were picked into the solution, and the number of head movements of the nematodes within 30 seconds was recorded under a stereomicroscope. Each group consisted of 15 nematodes, and the results were repeated 3 times.

[0048] Cardiac function indicators: In the late L4 stage, the number of pharyngeal pump beats of 15 nematodes per 30 seconds was recorded under a stereomicroscope, with 3 repetitions.

[0049] Reproductive capacity indicators: One L4-stage hermaphroditic nematode from each group was transferred to a new culture dish coated with OP50. The number of eggs laid was counted every 24 hours, and the nematode was transferred to a new culture medium until egg laying stopped. The total number of eggs laid was counted, and the experiment was repeated 6 times.

[0050] Lifespan indicators: Nematodes were cultured on NGM medium containing FUDR. Survival was observed and recorded every two days, starting from day 1 of adulthood. No reaction upon lightly touching the head of a nematode was considered death. Nematodes that died naturally, burrowed into the agar, or burst were removed from the experiment until all nematodes were dead. Survival curves were plotted, with 30 nematodes per group, repeated 3 times.

[0051] 3. Results The measurement of body length, body width, pharyngeal pump frequency, reproductive capacity, and lifespan was conducted to comprehensively evaluate the GCM1101 hybrid nematode from the dimensions of growth and development, feeding function, reproductive potential, and survival ability, and to establish a stable mitochondrial homeostasis model. Phenotypic and physiological status analysis of four nematode strains (N2, MQD753, ATU3301, and GCM1101) revealed that, among developmental-related indicators, the N2 wild-type nematode had a significantly longer body length than the other three strains (Figure 4, left) (P<0.001), and a significantly wider body width. Figure 4 (Right Figure) (P<0.01), but there were no significant differences in body length and width among MQD753, ATU3301, and GCM1101, indicating that GCM1101 nematode has no effect on development. Regarding locomotor function, the number of head swings in N2 was significantly higher than in MQD753 (P<0.001), because MQD753 exhibits a rolling phenotype, which has some influence on head swing. However, the number of head swings in GCM1101 and N2 nematodes was significantly increased (P<0.01), indicating that hybridization improved their locomotor ability to some extent (Figure 5, Left Figure). Since the pharynx of *C. elegans* is the organ for transporting food and can be considered a homologous organ to the vertebrate heart, its cardiotoxicity was evaluated by the number of pharyngeal pump beats. There were no significant differences in the pharyngeal pump frequency between GCM1101 and MQD753 and ATU3301, indicating that GCM11101 has no cardiotoxicity (Figure 5, Middle Figure). The oviposition rates of the four nematode strains (Figure 5, right) did not show significant differences; survival curves ( Figure 6 The results showed that N2, MQD753, ATU3301, and GCM1101 all died within approximately 25 days, with no significant difference in survival time. In conclusion, GCMM101 does not have serious genetic defects and is a stable model of mitochondrial homeostasis.

[0052] Example 4: Comparison and verification of staining methods between GCM1101 strain and wild type To visually demonstrate the unique advantages of the GCM1101 strain in monitoring mitochondrial superoxide (ROS) and calcium ions, this example compares its detection results with those of wild-type N2 nematodes using exogenous fluorescent dye staining.

[0053] 1. Medicines and reagents Reactive oxygen species detection kit: catalog number S0033S, purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Mitochondrial calcium ion detection kit (Rhod-2 AM): catalog number S10625, purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0054] 2. Methods Experimental groups: N2 staining group: ROS and mitochondrial calcium ion probe were used for staining; GCM1101 group: no staining was performed, and its intrinsic fluorescence was observed directly.

[0055] The synchronized N2 and GCM1101 strains were cultured to the L4 stage under the same conditions. Nematodes were collected after multiple washes with M9 buffer. In the N2 group, 500 μl of Rhod-2 staining solution (1X) was prepared according to the mitochondrial calcium ion instructions, followed by the addition of 1 μL of 2',7'-diacetic acid dichlorofluorescent resin (DCFH-DA). The nematodes were incubated together at 20°C for 3 hours in complete darkness. The probe was then washed away with M9 to avoid an overly bright background color. Finally, the nematodes were anesthetized with 0.5% levamisole hydrochloride and observed and images were acquired under a fluorescence inverted microscope (excitation wavelength of reactive oxygen species: 488 nm, emission wavelength: 525 nm; excitation wavelength of mitochondrial calcium ions: 552 nm, emission wavelength: 581 nm).

[0056] 3. Results Figure 7 As shown, after exogenous staining with MitoSOX and Rhod-2 / AM, the N2 wild-type nematodes showed increased levels of GFP channels (labeling mitochondrial ROS) and Cy3 channels (labeling mitochondrial Ca2+). 2+ Fluorescent signals could be detected in all of them. However, the signal distribution was uneven, background interference was significant, and the mitochondrial localization was not clear enough, making it difficult to accurately distinguish between mitochondrial-specific signals and non-specific background staining. Compared with the N2 staining group, the GCM1101 strain, without the need for exogenous staining, showed clear cpYFP (superoxide probe) green fluorescence in the mitochondria of the pharyngeal muscle and LAR-GECO (calcium ion probe) red fluorescence in the mitochondria of the body wall muscle. The signals were stable, the localization was accurate, and the background was clean. More importantly, it is convenient and can achieve simultaneous, real-time, and dynamic monitoring of dual signals in the same live nematode, eliminating problems such as dye toxicity, differences in loading efficiency, interference from washing steps, and signal quenching.

[0057] In summary, this invention provides a method for culturing *C. elegans* with dual fluorescent labeling for mitochondrial homeostasis monitoring through a hybridization strategy. The proposed strain integrates a mitochondrial superoxide sensor (cpYFP) and a mitochondrial calcium ion indicator (LAR-GECO) into a single in vivo model, enabling simultaneous real-time monitoring of two key mitochondrial homeostasis indicators. Through rigorous hybridization screening and multi-generational genetic stability verification, both the genotype and phenotype are stably inherited, ensuring the reliability, reproducibility, and consistency of experimental results. This strain requires no exogenous staining agents, avoiding problems such as dye toxicity, uneven penetration, rapid quenching, and strong background interference. The fluorescent protein is stably integrated into the genome and specifically located in mitochondria, providing stable signals, precise localization, and high reproducibility. This makes it highly suitable for constructing high-throughput drug screening platforms. This strain can be used to discover drugs or compounds that can regulate mitochondrial function and alleviate oxidative stress and calcium overload. It provides a novel tool with physiological relevance, ease of operation, and cost advantages for mitochondrial homeostasis mechanism research and related disease drug development, possessing significant potential application value.

[0058] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing *C. elegans* with dual fluorescent labels for mitochondrial homeostasis monitoring, characterized in that, Includes the following steps: (1) Cultivate the ATU3301 strain of Caenorhabditis elegans to the L4 larval stage and select males as the male parent; (2) Cultivate the Caenorhabditis elegans MQD753 strain to the L4 larval stage, and select hermaphroditic individuals as the female parent; (3) Place them on a culture plate in proportion, mate them, and obtain offspring; (4) Remove the parent and continue to raise the offspring to the L2 larval stage; (5) The target Caenorhabditis elegans was obtained by screening Caenorhabditis elegans under a microscope.

2. The cultivation method as described in claim 1, characterized in that, The ratio mentioned in step (3) is 1:1 to 1:

3.

3. The cultivation method as described in claim 1, characterized in that, The culture plate mentioned in step (3) is an NGM culture plate.

4. The cultivation method as described in claim 1, characterized in that, The microscope screening described in step (5) includes stereomicroscope screening and fluorescence microscope screening.

5. The cultivation method as described in claim 4, characterized in that, The stereomicroscopy screening method is used to screen nematodes with a rolling phenotype. The fluorescence microscopy screening method is based on the stereomicroscopy screening method and selects nematodes whose pharyngeal muscles emit both cpYFP green fluorescence and RFP red fluorescence, and whose body walls have LAR-GECO red fluorescence.

6. The cultivation method as described in claim 1, characterized in that, The target Caenorhabditis elegans genome described in step (5) is stably integrated with the hqIs180 [sdhb-1p::mtLS::cpYFP+rol-6(su1006)] genetic element from the MQD753 strain and the aceIs1 [myo-3p::mitochondrial LAR-GECO + myo-2p::RFP] genetic element from the ATU3301 strain.

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