LU505264B1 - Establishment Method of a Tumor Invasion Model in Drosophila melanogaster - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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Abstract
Disclosed is a method for constructing a fruit fly tumour invasion model. The method involves hybridizing virgin female fruit flies with male fruit flies. With this method, the Dorsal.H; RasV12 tumour invasion model in black abdomen fruit flies has been successfully established. Utilizing this model, researchers can conduct screenings within the entire fruit fly genome to identify gene mutations or RNAi that inhibit tumour migration, elucidate the molecular mechanisms regulating tumour migration, and perform functional validation of homologous genes in mammals. Additionally, large-scale drug screening of chemical compounds, traditional Chinese medicine formulations, individual herbal medicines, or monomers can be carried out, aiming to provide new drugs, drug targets, and treatment approaches for clinical cancer therapy.
Description
Establishment Method of a Tumor Invasion Model in Drosophila melanogaster
LU505264
This invention belongs to the field of genetic biology technology and specifically relates to a method for establishing a tumour invasion model of Drosophila melanogaster.
Existing Drosophila Genetic Operating Systems include the GAL4/UAS Dual Expression
System and the FLP/FRT System. Among these, (1) the GAL4/UAS Dual Expression System is the most commonly used transgenic technology system in Drosophila, allowing for selective expression of exogenous genes or RNAI in specific cells or tissues. The system is based on the
Galactose-Regulated Upstream Promoter Element 4 (GAL4), an activator transcription factor similar to prokaryotic lactose operons found in yeast. The Upstream Activating Sequence (UAS) is another sequence in yeast that functions similarly to enhancers in higher eukaryotes. GAL4 regulates the expression of genes related to galactose metabolism by binding to UAS. An illustration of the GAL4/UAS Dual Expression System is shown in Figure 1. In 1993, scientists linked the target gene X downstream of UAS and established UAS-X Drosophila strains using transgenic technology. These strains were then crossed with specific GAL4 Drosophila strains to obtain offspring with both Y-Gal4 and UAS-X, thereby achieving specific expression of the X gene — in tissues (Reference 1, Brand A. H. and Perrimon N., Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, 1993, 118: 401-15).
Since the Drosophila genome does not encode the GAL4 transcription factor, overexpressing
GAL4 within Drosophila does not significantly affect its development. Similarly, inserting the UAS regulatory sequence within Drosophila has no impact on the organism. The establishment of this system has provided scientists using Drosophila as a research model with a convenient and efficient genetic manipulation tool, facilitating experimental design. (2) FLP/FRT System: FLP is a recombinase enzyme found in yeast, which can recognize two homologous 700bp target sites known as FRTs (FLP recombination targets). If two FRT segments are located at the same position on a pair of homologous chromosomes in Drosophila, heat- induced FLP expression can mediate mitotic recombination between these two sites, generating recombinant distal homozygous daughter cells (as shown in Figure 2) (Reference 2, Zhang S. P. and Xue L., [Progress on cell lineage analysis in Drosophila melanogaster]. Yi Chuan, 2012, 34: 819-28). Studies have shown that FRT-mediated mitotic recombination is far more efficient than other methods, and the sites of recombination can be controlled artificially. Furthermore, heat- induced damage to cells is minimal. Therefore, researchers can use the FLP/FRT technique to investigate the growth of cells with minor genetic alterations in a wild-type cellular environment, further exploring the molecular mechanisms of cell competition.
Drosophila, as a model organism for the study of human diseases, shares not only fundamental biological, physiological, and neurological similarities with mammals but also offers 05264 unique advantages as a model organism. Recent research has demonstrated a high degree of conservation between Drosophila and humans in signalling pathways related to tumorigenesis.
Additionally, Drosophila possesses robust genetic manipulability, making it an effective model for studying the molecular mechanisms underlying cancer initiation, progression, and metastasis. In recent years, researchers have established numerous Drosophila models tailored for specific studies. However, the establishment of novel, specific Drosophila tumour invasion models is of paramount importance for elucidating the molecular mechanisms underlying diseases such as cancer. Such models hold the potential to provide new drug targets and treatment strategies for clinical therapy. Therefore, the creation of novel, specific Drosophila tumour invasion models represents a pressing technical challenge for professionals in this field.
The objective of this invention is to provide a method for establishing a tumour invasion model of Drosophila melanogaster.
The objective of this invention is achieved through the following technical procedure:
A method for establishing a tumour invasion model of Drosophila melanogaster with black abdomens, wherein the method involves hybridizing collected virgin female flies and healthy male flies according to a hybridization process. The hybridization process comprises the following steps: (1) Hybridize Drosophila strains UAS-Dorsal.H and Sp/Cyo; Sb/TM6B.Tb, selecting male flies with the genotype UAS-Dorsal.H/Sp; +/Sb in the offspring, denoted as 'a. Simultaneously, hybridize Drosophila strains Sp/Cyo; Sb/TM6B.Tb and UAS-RasV'?, selecting female flies with the genotype +/Cyo; UAS-Ras”"?/TM6B.Tb, denoted as 'b.' (2) Hybridize 'a' and 'b," selecting male flies in the offspring with the genotype UAS-
Dorsal.H/Cyo; UAS-Ras"'?/Sb, denoted as 'c.' Hybridize 'c' with female flies of the Sp; Sb/SM6B-
TM6B.Tb strain, selecting flies with the genotype UAS-Dorsal.H; UAS-Ras"'?/SM6B-TM6B. Tb, establishing a Drosophila strain that can be propagated and preserved. (3) The Drosophila strain in which GFP-marked eye tissue tumour cells have migrated to the abdominal ganglia is identified as the Dorsal.H; Ras"? tumour migration model in Drosophila with black abdomens.
As a preferred technical approach, the detailed process for step (2) is as follows: Hybridize the ey-Flp act>y+>GAL4 UAS-GFP strain (abbreviated as ey-Flp, GFP) with the 'd' obtained in step (1). In the offspring, select third-instar larvae with the genotype ey-Flp, GFP/UAS-Dorsal.H;
UAS-RasV'?/+. These larvae exhibit the phenotype of GFP expression in the Cephalic Complex (CC) region and have a short, stocky body. After dissecting the two brain hemispheres and the
Ventral Nerve Cord (VNC) of the Drosophila, observe that GFP-labelled tumour cells in the eye tissue have migrated to the Drosophila's abdominal ganglia. This confirms the establishment of the Dorsal.H; Ras"? tumour migration model in Drosophila.
LU505264
As a further preferred approach: When hybridizing the ey-Flp act>y+>GAL4 UAS-GFP
Drosophila strain with the 'd' obtained in step (1), the parental male and female used for hybridization can be interchangeable.
As a preferred technical approach: The formulation of the culture medium for the hybridization is as follows: 24.21% sucrose, 0.96% agar, 12.38% cornmeal, 0.57% yeast, 0.32% propionic acid.
As a preferred technical approach: Cultivate under conditions with a temperature of 22°C and humidity between 50% to 70%.
The method for selecting virgin female flies is as follows: Remove all adult flies from the stock vial, then collect recently eclosed virgin female flies every 8 hours and place them in a culture vial for later use. Because newly eclosed flies are slender, delicate, and nearly transparent, their abdomens have a chitin exoskeleton that allows observation of the black digestive tract inside the abdominal cavity. Therefore, female individuals with a visible black digestive tract are considered virgin flies.
The beneficial effects of the present invention:
The method successfully establishes the Dorsal.H; Ras”? tumour invasion model in
Drosophila. Using this model, researchers can conduct genome-wide screenings in Drosophila to identify gene mutations or RNAI that inhibit tumour migration. This helps elucidate the molecular mechanisms regulating tumour migration. Additionally, functional validation of homologous genes can be performed in mammals. Large-scale drug screenings for synthetic compounds, traditional
Chinese medicine formulas, single herbs, or single compounds can also be conducted, aiming to provide new drugs, drug targets, and treatment strategies for clinical cancer therapy.
Figure 1 depicts a schematic diagram of the GAL4/UAS Dual Expression System.
Figure 2 illustrates a schematic representation of the FLP/FRT System's operating principle.
Figure 3 provides a schematic representation of Drosophila sexual dimorphism.
Figure 4 presents a flowchart outlining the hybridization process for constructing the Dorsal.H;
Ras“? tumour invasion model.
Figure 5 demonstrates that co-expression of Dorsal and Ras can induce tumour invasion.
Based on the specific examples described below, a detailed description of the present invention is provided. From these descriptions and examples, those skilled in the art can discern the fundamental characteristics of the present invention. Furthermore, it is understood that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, to adapt it for various applications and conditions.
Example LU505264 (a) Drosophila Rearing and Experimental Conditions 1. Preparation of Drosophila Culture Medium**
Drosophila used in the experiments, as well as the hybridization experiments, were cultured on a standard medium composed of sucrose, cornmeal, and yeast. The formulation of the culture medium is as follows:
Brown sugar: 135 g
Agar: 74
Corn starch: 85g
Yeast: 89g
Propionic acid: 4 mi
Dilute with water to volume: 1000 ml
Preparation Procedure: (1) Combine the pre-weighed sucrose and agar in an electric rice cooker, add an appropriate amount of water, and stir thoroughly. (2) Heat the mixture until it reaches a boiling point. (3) Slowly pour the pre-dissolved cornmeal into the pot while stirring continuously. (4) Heat the mixture until it reaches a boiling point again. (5) Allow the mixture to cool to around 80°C, then add the pre-dissolved yeast, which is the high-activity dry yeast produced by AB/MAURI™ under the brand name "Meishan Highly
Active Dry Yeast," 500g/package. Stir thoroughly. (6) Add an appropriate amount of propionic acid solution and stir well. (7) Dispense the culture medium into sterilized glass tubes. (8) Plug the tubes with cotton and store them in a cool place. 2. Experimental Conditions
Maintain a constant temperature of 25°C and humidity between 50% and 60%.
Hybridization experiments are typically conducted in a climate-controlled incubator or a fruit fly room with constant temperature and humidity. 3. Differentiation of Female and Male Drosophila (1) Body Size: Female flies have larger body sizes compared to males. (2) Abdominal Tip: Female flies have slightly pointed elliptical abdominal tips, while male flies have blunt and round tips. (3) Abdominal Dorsal Side: Female flies exhibit five distinct black stripes on their dorsal abdominal side, whereas male flies have three stripes. The first two stripes are narrow, and the third one is wider and extends to the ventral side, with a visible black dot at the tip.
(4) Abdominal Ventral Side: Female flies have six noticeable abdominal segments, while male 505264 flies have four. (5) Sex Comb: Male flies possess a black hair-like structure called a sex comb on the upper front surface of the first tarsal segment of their legs. 5 (6) Genitalia: The primary distinguishing feature between male and female flies.
A schematic representation of the differentiation between male and female Drosophila is provided in Figure 3. 4. Anaesthesia and Hybridization of Drosophila
The anaesthesia method for Drosophila involves the use of carbon dioxide (CO2) gas.
When using parent Drosophila for hybridization experiments, it is crucial not to over-anesthetize them, as it can affect the fly's vitality. The differentiation between the anesthetized state and post-anaesthesia mortality is based on the position of the wings. Anesthetized flies have both wings overlapping on their dorsal side, while dead flies have wings extended away from their abdomen.
Once the flies are anesthetized, various operations such as sex selection, hybridization, and phenotypic observation can be performed on a CO--saturated platform. Since female fruit flies have a seminal receptacle in their reproductive organs, capable of storing a significant amount of sperm after a single mating for multiple ovipositions, it is essential to collect virgin flies that have not mated before for reliable hybridization experiments. The selection method involves removing all adult flies from the stock vial and collecting recently eclosed female fruit flies every 8 hours, placing them in culture vials for future use. Newly eclosed fruit flies have a slender and delicate body, almost transparent, with a visible black digestive tract through the chitinous exoskeleton on the ventral side of the abdomen. Therefore, females with a visible black digestive tract are considered virgin flies. (b) A Method for Establishing a Tumor Invasion Model in Drosophila melanogaster. 1. Fruit Fly Strains Required for Model Establishment (1) w°1"8, wild-type fruit flies, obtained from the Bloomington Drosophila Stock Center in the
United States, catalogue number BL3605. (2) ey-Flp act>y+>GAL4 UAS-GFP, located on the second chromosome, cannot be homozygous, with the specific genotype: w°°78; ey-Flp act>y+>GAL4 UAS-GFP/Cyo, purchased from the Fruit Fly Resource and Technology Platform at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. (3) UAS-Dorsal.H, a wild-type Dorsal strain, located on the second chromosome, with yellow- eyed fruit flies that can be homozygous, with the specific genotype: w°"78: UAS-Dorsal.H, obtained from the Bloomington Drosophila Stock Center in the United States, catalogue number BL9319.
(4) UAS-Ras''?, located on the third chromosome, can be homozygous, with the specific LU505264 genotype: w; UAS-Ras"'?, purchased from the Bloomington Drosophila Stock Center in the
United States, catalogue number BL64159. (5) Sp/Cyo; Sb/TM6B.Tb, a second and third chromosome balancer tool fruit fly strain, obtained from the Fruit Fly Resource and Technology Platform at the Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences. (6) Sp; Sb/SM6B-TM6B.Tb, a tool fruit fly strain with a second-third chromosome linkage, purchased from the Fruit Fly Resource and Technology Platform at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. 2. The Resulting Genotypes are as Follows:
Figure 5A: ey-Flp act>y+>GAL4 UAS-GFP/+
Figure 5B: ey-Flp act>y+>GAL4 UAS-GFP/+; UAS-RasV'?/+
Figure 5C: ey-Flp act>y+>GAL4 UAS-GFP/UAS-Dorsal.H; UAS-Ras"!?/+
Figure 5D: ey-Flp act>y+>GAL4 UAS-GFP/UAS-Dorsal.H 3. Experimental Method (1) Hybridization of Collected Virgin Female Flies and Healthy Male Fruit Flies in accordance with the hybridization process, as depicted in Figure 4. The detailed steps are as follows:
Cross the UAS-Dorsal.H fruit fly strain with the Sp/Cyo; Sb/TM6B.Tb fruit fly strain. In the offspring, select male flies with the genotype UAS-Dorsal.H/Sp; +/Sb. Simultaneously, cross the Sp/Cyo; Sb/TM6B.Tb fruit fly strain with the UAS-Ras"'? fruit fly strain. In the offspring, select female flies with the genotype +/Cyo; UAS-Ras‘"?/TM6B.Tb. Then, cross the obtained UAS-Dorsal.H/Sp; +/Sb male flies with +/Cyo; UAS-Ras/"?/TM6B.Tb female flies. In the offspring, select male flies with the genotype UAS-Dorsal.H/Cyo; UAS-
RasV'?/Sb. (2) Subsequently, cross the obtained UAS-Dorsal.H/Cyo; UAS-Ras"'?/Sb male flies with female flies from the Sp; Sb/SM6B-TM6B.Tb strain. In the offspring, select fruit flies with the genotype UAS-Dorsal.H; UAS-Ras‘"?/SM6B-TM6B.Tb, with an appropriate ratio of males and females, and establish a fruit fly strain that can be propagated and preserved. (3) Collect the target fruit flies from the hybridized offspring and dissect the heads of third-instar fruit fly larvae, along with the abdominal ganglia. Place these dissected tissues under a stereomicroscope equipped with a fluorescence imaging system and capture photographs. 4. Model Overview
Figures 5A - D depict dissected complex tissues of the cephalic complex (CC) in third-instar fruit fly larvae, with "EA" representing the eye-antennal discs, "BH" representing the two brain hemispheres of the fruit fly, and "VNC" representing the ventral nerve cord.
Using FLP-FRT-mediated mitotic recombination in conjunction with the GAL4/UAS system, 505264 we observed that compared to the control group (Figure 5A, A"), the specific continuous expression of the highly activated oncogene Ras (Ras"'?) in the eye-antennal discs could promote in situ overgrowth of tumour cells (Figure 5B, B"). Furthermore, co-expression of the human NF-kB homologous factor, Dorsal, synergistically induced tumour growth, and these tumour cells invaded the ventral nerve cord (tumour cells were labelled with GFP green fluorescent protein, Figure 5C, C'). In contrast, expressing Dorsal alone did not result in the invasive phenotype (Figure 5D, D"). This indicates the successful establishment of the Dorsal. H;
Ras"? tumour invasion model (Figure 5 demonstrates that co-expression of Dorsal and Ras induces tumour invasion).
Researchers can utilize this model to conduct genome-wide screens in Drosophila, identifying gene mutations or RNAI that inhibit tumour migration. This helps elucidate the molecular mechanisms regulating tumour migration. Furthermore, functional validation of homologous genes can be performed in mammals. Large-scale drug screening of chemical compounds, traditional Chinese medicine formulations, individual herbal medicines, or monomers can also be carried out using this model. This approach aims to provide new drugs, drug targets, and treatment strategies for clinical cancer therapy.
Claims (6)
1. A method for establishing a Drosophila melanogaster tumour invasion model, comprising the following steps: (1) hybridizing virgin female flies and male fruit flies, with the hybridization process comprising the following steps: (a) - crossing UAS-Dorsal.H and Sp/Cyo; Sb/TM6B.Tb Drosophila strains; — selecting in the offspring male fruit flies with the genotype UAS-Dorsal.H/Sp; +/Sb, denoted as "a." — simultaneously, crossing the Sp/Cyo; Sb/TM6B.Tb fruit fly strain with the UAS- Ras“? fruit fly strain; — selecting in the offspring female fruit flies with the genotype +/Cyo; UAS- RasV'?/TM6B.Tb, denoted as "b." (b) - crossing "a" and "b."; — selecting in the offspring male fruit flies with the genotype UAS-Dorsal.H/Cyo; UAS-Ras/?/Sb, denoted as "c."; — crossing "c" with female fruit flies from the Sp; Sb/SM6B-TM6B.Tb strain; — selecting in the offspring fruit flies with the genotype UAS-Dorsal.H; UAS- Ras/2/SM6B-TM6B.Tb, capable of being propagated and preserved. (2) taking the fruit fly strains with GFP-labelled eye tissues that have migrated to the abdominal ganglia as the Dorsal.H; Ras"? tumour migration model for fruit flies.
2. The method for establishing the Drosophila melanogaster tumour invasion model according to claim 1, wherein in step (2) comprises — crossing the ey-Flp act>y+>GAL4 UAS-GFP fruit fly strain with the "d" obtained in step (1); — selecting in the offspring third-instar larvae with the genotype ey-Flp, GFP/UAS- Dorsal.H; UAS-Ras"'?/+, exhibiting a phenotype with GFP-labelled eye tissues and a stout body; — dissecting the fruit fly and confirming, by observing both brain hemispheres and the abdominal ganglia, the migration of GFP-labelled tumour cells from the eye tissues to the fruit fly's abdominal ganglia, therewith establishing the Dorsal. H; Ras"? tumour migration model for fruit flies.
3. The method for establishing the Drosophila melanogaster tumour invasion model according to claim 2, wherein the parental male and female flies used for the cross in step (2) are not interchangeable.
4. The method for establishing the black abdomen Drosophila melanogaster tumour invasion 505264 model according to claim 1, wherein the composition of the hybridization medium comprises
24.21% brown sugar, 0.96% agar, 12.38% corn starch, 0.57% yeast, 0.32% propionic acid.
5. The method for establishing the Drosophila melanogaster tumour invasion model according to claim 1, wherein the rearing conditions comprise culturing at a temperature of 22°C and humidity between 50% - 70%.
6. The method for establishing the Drosophila melanogaster tumour invasion model according to claim 1, the method of selecting virgin female flies comprising: — clearing all adult flies from the original vial, — then collecting recently eclosed female fruit flies every 6 hours, — selecting virgin female flies from the newly eclosed fruit flies by considering female individuals with a visible black digestive tract as virgin flies, based on the observation that newly eclosed fruit flies have slender, nearly transparent bodies, and a black digestive tract can be seen through the chitinous exoskeleton on the ventral side of the abdomen; — placing the selected virgin female flies in culture vials for future use.
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