CN108184770B - Drosophila melanogaster RasV12Method for establishing Snail tumor migration model - Google Patents
Drosophila melanogaster RasV12Method for establishing Snail tumor migration model Download PDFInfo
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Abstract
The invention discloses drosophila melanogaster RasV12Snail tumor migration modelThe establishment method comprises the steps of adopting a specific drosophila strain to carry out hybridization, culturing and screening to obtain drosophila melanogaster RasV12A Snail tumor migration model; drosophila melanogaster Ras constructed by using method of the inventionV12A study person can use the model to carry out large-scale genetic screening, search gene mutation or RNAi for inhibiting tumor migration, clarify a molecular mechanism for regulating and controlling tumor migration, and carry out functional verification on homologues of the tumor in mammalian cells so as to provide a new drug target and a new treatment scheme for clinical treatment of cancer.
Description
Technical Field
The invention belongs to the technical field of biological genetics, and particularly relates to drosophila melanogaster RasV12A method for establishing a Snail tumor migration model is provided.
Background
Drosophila genetics operating systems include Gal4/UAS double expression system and FLP/FRT system. Wherein, the two expression systems of (I) Gal 4/UAS: the Gal4/UAS system is the most commonly used transgenic technology system in Drosophila, and allows the selective expression of exogenous genes or RNAi in specific cells or tissues. The galactose-regulated upstream promoter element (4), abbreviated Gal4, is a transcriptional activator in yeast similar to the prokaryotic lactose operon. Upstream activation sequenceUAS(upstream actual sequence) is another enhancer-like sequence in yeast. Gal4 is prepared by reacting withUASIn combination, the expression of the galactose metabolism-related gene is regulated. In 1993, scientists connected the target gene XUASThen, it is established by transgenic technologyUASthe-X Drosophila strain, which is then crossed with a specific Gal4 Drosophila strain, can be obtained in the offspring while havingY-Gal4 andUASdrosophila melanogaster of X, thereby achieving specific expression of the X gene in Y tissue (reference 1, Brand A.H. and Perrimon N., Targeted genetic expression as a means of alternative cells and genetic encoding phenyl)types.Development, 1993, 118: 401-15). Since the drosophila genome does not encode the Gal4 transcription factor, overexpression of Gal4 in drosophila does not have a significant effect on drosophila development. Also, inserted into the body of DrosophilaUASThe regulatory sequence can not influence Drosophila. The establishment of the system provides a favorable, convenient and efficient genetic operation tool for scientists using fruit flies as research models in experimental design. A schematic diagram of the Gal4/UAS dual expression system is shown in FIG. 1.
(II) FLP/FRT system: FLP is a recombinase in yeast that recognizes two 700bp homologous target sites FRT (FRTs), and if two FRT fragments are located at the same site on a pair of homologous chromosomes in Drosophila, the expression of FLP induced by heat shock can mediate mitotic recombination at these two sites to generate recombinant distant homozygous daughter cells (FIG. 2) (reference 2, Zhang S.P. and Xue L., [ Progress on cell lineageanalysis in Drosophila melanogaster)].Yi Chuan, 2012, 34: 819-28). It was found that FRT-mediated mitotic recombination is much more efficient than other means and that the site at which recombination occurs can also be artificially controlled. In addition, the heat shock is less harmful to the cells. Therefore, researchers can explore the growth of a few genetically altered cells in the environment of wild-type cells by the FLP/FRT technique, thereby further investigating the molecular mechanisms of cell competition. The FLP/FRT system is schematically shown in FIG. 2.
The drosophila, as a model organism for studying human diseases, is not only similar to mammals in terms of basic biology, physiology, nervous system function and the like, but also has unique advantages of the drosophila as the model organism. Recent researches show that fruit flies and human beings have high conservation in the aspects of tumorigenesis signal pathways and the like, and the fruit flies have strong genetic operability, are one of effective models for oncology researches, and can be used for researching molecular mechanisms of human tumorigenesis, development, metastasis and the like. Researchers have established many drosophila models for studying specific cancers in recent years, but the establishment of new drosophila models for specific cancers plays an important role in elucidating the molecular mechanisms of tumorigenesis, and will provide new drug targets and treatment protocols for the clinical treatment of more cancers. Therefore, establishing a new drosophila model of a specific tumor is a technical problem to be solved urgently by those skilled in the art.
Disclosure of Invention
The invention aims to provide drosophila melanogaster RasV12A method for establishing a Snail tumor migration model and applications thereof are provided.
Another object of the present invention is to provide drosophila melanogaster Ras established by the above methodV12A Snail tumor migration model.
The purpose of the invention can be realized by the following technical scheme:
drosophila melanogaster RasV12The method for establishing the Snail tumor migration model comprises the steps of hybridizing specific drosophila melanogaster strains, culturing and screening to obtain drosophila melanogaster RasV12A Snail tumor migration model; the method specifically comprises the following steps:
(1) will be provided withUAS-Snail74bThe drosophila strain is hybridized with Sp/Cyo and Sb/TM6B.Tb drosophila strain, and the genotype is selected from the progenyUAS-Snail74b/Sp;+Male fruit flies of Sb; simultaneously, Sp/Cyo, Sb/TM6B.Tb drosophila strains are combinedUAS-RasV12The fruit fly strains are hybridized, and the genotype is selected from the offspring+/Cyo;UAS-RasV12 /Tm6b.tb female drosophila; then will obtainUAS-Snail74b/Sp;+Male drosophila/Sb and +/Cyo, UAS-RasV12Female drosophila/TM6B.Tb is hybridized, and the genotype is selected from the progenyUAS-Snail74b/Cyo;UAS-RasV12Male fruit flies of Sb; then, will obtainUAS-Snail74b/Cyo;UAS-RasV12Hybridizing male Sb drosophila with female drosophila of Sb/SM6B-TM6B.Tb strain with genotype Sp and selecting genotype asUAS-Snail74b;UAS-RasV12the/SM 6B-TM6B.Tb drosophila melanogaster with proper male and female parts is built into a Stock strain;
(2) will be provided withey-Flpact>y+>Gal4UASGFP (abbreviated toeyFlp, GFP) Drosophila strains and final harvest in step (1)Obtained byUAS-Snail74b;UAS-RasV12the/SM 6B-TM6B.Tb drosophila strain is hybridized, and the genotype is selected from the offspringey-Flp, GFP/UAS-Snail74b;UAS-RasV12The third instar larva with GFP phenotype and non-squash body type is drosophila melanogaster RasV12A Snail tumor migration model.
In the step (1)UAS-Snail74bThe drosophila strain and Sp/Cyo, Sb/TM6B.Tb the drosophila strain can be combined with parents in any way when the drosophila strain is hybridized; Sp/Cyo, Sb/TM6B.Tb Drosophila strain andUAS-RasV12the male and female parents can be combined randomly when the fruit fly strains are hybridized; in the step (2)ey-Flpact>y+>Gal4UASGFP Drosophila strains and finally obtained in step (1)UAS-Snail74b;UAS-RasV12The parents can be combined in any way when the/SM 6B-TM6B.Tb drosophila strain is hybridized.
The formula of the culture medium used in the culture process comprises 135g of brown sugar, 7g of agar, 85g of corn flour, 8g of yeast, 4ml of propionic acid and 1000ml of water.
The culture conditions are as follows: the temperature is constant at 20-30 ℃ (preferably 25 ℃), and the humidity is 50% -60%.
The process of crossing the drosophila strain is as follows: anaesthetizing fruit fly with CO2The operation of selecting male and female, hybridizing and observing phenotype is carried out on the flat plate; virgin flies which have not been mated must be collected before hybridization experiments are carried out; the method for selecting the virgins comprises the steps of completely removing the imagoes in the stock bottle, and collecting the female fruit flies which are just eclosion every 8 hours, namely the virgins.
The method is used for constructing drosophila melanogaster RasV12Application in Snail tumor migration model.
The drosophila melanogaster Ras constructed by the methodV12A Snail tumor migration model.
The invention has the beneficial effects that:
drosophila melanogaster Ras constructed by using method of the inventionV12Snail tumor migration model, which researchers can use to carry out large-scale genetic screening to find out the gene inhibiting tumor migrationDue to mutation or RNAi, the molecular mechanism of regulating tumor migration is clarified, and the homolog of the mutant is functionally verified in mammalian cells, so as to provide a new drug target and a new treatment scheme for clinical treatment of cancer.
Drawings
FIG. 1 is a schematic diagram of a Gal4/UAS dual expression system.
FIG. 2 is a schematic diagram of the FLP/FRT system.
FIG. 3 is a schematic diagram showing fruit fly sex discrimination.
FIG. 4 is a schematic representation of the hybridization procedure for Drosophila melanogaster.
FIG. 5 shows that increasing Snail promotes tumor growth and RasV12Migration of the cells.
In which panels A-D are head complex tissue (Cephalic complex) anatomies of the third instar larvae stage of Drosophila melanogaster, in which EA represents the eye-insect disc (eye-anti disc), BH represents the two cerebral hemispheres of Drosophila melanogaster, and VNC represents the Ventral ganglion (Ventral nerve cord, VNC).
Detailed Description
Example 1 establishment of Drosophila melanogaster RasV12Snail tumor migration model
Feeding and experimental conditions of drosophila melanogaster
Preparation of culture medium for fruit fly
Experimental drosophila strains and hybridization experiments drosophila were raised on a standard brown sugar-corn flour-yeast medium, wherein the medium formulation was as follows: 135g of brown sugar, 7g of agar, 85g of corn flour, 8g of yeast, 4ml of propionic acid and 1000ml of water.
The preparation process comprises the following steps: (1) pouring the weighed brown sugar and agar into an electric cooker, adding a proper amount of water, and fully stirring; (2) heating to boiling; (3) slowly pouring the corn flour which is fully dissolved by water into a pot, and continuously stirring; (4) heating to boiling; (5) cooling the mixture to about 80 deg.C, adding yeast dissolved in warm water in advance, and stirring thoroughly; (6) adding a proper amount of propionic acid solution, and fully stirring; (7) subpackaging the culture medium into sterilized glass tubes; (8) after cotton is filled in the cotton plug, the cotton plug is placed in a shade place for storage.
(II) Experimental conditions
The temperature is kept at 25 ℃, the humidity is 50-60%, and the fruit flies are generally cultured in an incubator or a fruit fly room with the temperature and the humidity being 25 ℃.
(III) identification of female and male fruit flies
(1) Body type: female drosophila are larger in size and male ones are smaller. (2) Abdominal end: the oval end of the abdomen of the female drosophila is slightly sharp, and the end of the male drosophila is blunt and round. (3) Back of abdomen: the female fruit flies have 5 obvious black stripes, the male fruit flies have 3 stripes, the first 2 stripes are thin, the second 1 stripe are wide, and the tail end of the abdomen is visible to naked eyes to have an obvious black spot. (4) Abdomen and abdomen surface: female fruit flies have 6 obvious belly patches, and male fruit flies have 4 belly patches. (5) Sex comb: the front end surface of the foot attached to the uppermost part of the sole side of the first segment of the male fruit fly is provided with a black bristle-sexual comb. (6) A tail-crossing device: the most important difference of male and female fruit flies is judged. A schematic diagram of fruit fly sexing is shown in FIG. 3.
(IV) anesthesia and hybridization of Drosophila
The method for anesthetizing fruit flies is carbon dioxide (CO)2) Gas anesthesia method. The parent fruit fly used for hybridization experiment can not be overnarcotized, otherwise, the vitality of the fruit fly can be influenced. The distinction between the anesthetic state of drosophila and death after anesthesia depends on whether the wings are abducted. The two wings of the anesthetized drosophila are still overlapped on the dorsoventral, while the dead drosophila wings are spread out away from the ventral.
Placing fruit fly in the container with CO2The fruit flies can be anesthetized on the flat plate for 3 to 5 seconds. After the drosophila is anesthetized, according to the difference of appearance phenotypes of female drosophila and male drosophila, collecting unmatched female virginator flies and healthy male drosophila respectively, combining parents (female flies and male flies) required by hybridization according to a hybridization experiment flow, placing the parents in the same drosophila tube (with fresh culture medium added) for feeding, collecting offspring, and observing the phenotype of the hybridization target offspring.
Because the female drosophila reproductive organ has the seminal vesicle, a large amount of sperms can be stored after one-time mating for multiple ovulation and fertilization, so that virgin flies which are not mated must be collected before a hybridization experiment is carried out, otherwise, the experimental result is unreliable. The selection method comprises the following steps: all imagoes in the stock bottle are removed, and the female drosophila which is just eclosion is collected every 8 hours and put into a culture bottle for standby. The newly emerged fruit fly, which is slender and tender and almost transparent, penetrates the shell of chitin from the ventral surface of the abdomen and sees the dark digestive tract in the abdominal cavity. Therefore, the female individuals with black digestive tracts were seen as virgins.
Two, black drosophila RasV12Establishment of Snail tumor migration model
1. Fruit fly strain required for establishing model
（1）ey-Flpact>y+>Gal4UASGFP, located on drosophila melanogaster chromosome ii, cannot be homozygous, the specific genotype being:w 1118 ;ey-Flpact>y+>Gal4UASGFP/Cyo, ordered from Biochemical and cellular institute Drosophila resources and technology platform, Shanghai, Chinese academy.
（2）UAS-RasV12And the gene is positioned on the third chromosome of the drosophila, can be homozygous, and has the following specific genotypes:w;UAS-RasV12bloomington, subscribed to the university of Indiana, USADrosophilaStock Center, No.: BL 64159.
（3）UAS-Snail74bThe wild type Snail is positioned on the second chromosome of the drosophila melanogaster, the eye of the drosophila melanogaster is yellow and can be homozygous, and the specific genotype is as follows:w 1118 ;UAS-Snail74bordering Drosophila melanogaster resources and technical platform of Biochemical and cell institute of Shanghai academy of Chinese sciences.
(4) Sp/Cyo, Sb/TM6B.Tb, Drosophila melanogaster No. two chromosome tool, and is ordered to Drosophila melanogaster resource and technical platform of Biochemical and cell institute of Shanghai academy of China.
(5) Sp, Sb/SM6B-TM6B.Tb, Drosophila melanogaster No. two linked tools, and is ordered to Drosophila melanogaster resource and technology platform of Biochemical and cell institute of Shanghai academy of Chinese sciences.
（6）w 1118 Located on the drosophila X chromosome, ordered at the drosophila center of the university of qinghua, code: THJ 0265.
The resulting genotypes were as follows
FIG. 5A:ey-Flpact>y+>Gal4UAS-GFP/+
FIG. 5B:ey-Flpact>y+>Gal4UAS-GFP/+;UAS-RasV12/+
FIG. 5C:ey-Flpact>y+>Gal4UAS-GFP/UAS-Snail74b;UAS-RasV12/+
FIG. 5D:ey-Flpact>y+>Gal4UAS-GFP/UAS-Snail74b
3. experimental methods
(1) The collected female virgins and healthy male fruit flies are hybridized according to a hybridization process, the fruit fly hybridization process is shown in figure 4, and the detailed steps are as follows:
a. will be provided withUAS-Snail74bThe drosophila strain is hybridized with Sp/Cyo and Sb/TM6B.Tb drosophila strain, and the genotype is selected from the progenyUAS-Snail74b/Sp;+Male fruit flies of Sb; simultaneously, Sp/Cyo, Sb/TM6B.Tb drosophila strains are combinedUAS-RasV12The fruit fly strains are hybridized, and the genotype is selected from the offspring+/Cyo;UAS-RasV12 /Tm6b.tb female drosophila; then will obtainUAS-Snail74b/Sp;+Male drosophila/Sb and +/Cyo, UAS-RasV12Female drosophila/TM6B.Tb is hybridized, and the genotype is selected from the progenyUAS-Snail74b/Cyo;UAS-RasV12Male fruit flies of Sb; then, will obtainUAS-Snail74b/Cyo;UAS-RasV12Hybridizing male Sb drosophila with female drosophila of Sb/SM6B-TM6B.Tb strain with genotype Sp and selecting genotype asUAS-Snail74b;UAS-RasV12the/SM 6B-TM6B.Tb fruit fly has proper male and female, and can be used for establishing a fruit fly strain which can be subcultured.
b. Will be provided withey-Flpact>y+>Gal4UASGFP (abbreviated toeyFlp, GFP) Drosophila strains and finally obtained in step aUAS-Snail74b;UAS-RasV12the/SM 6B-TM6B.Tb drosophila strain is crossed and thenThe genotype is selected from the generation asey-Flp, GFP/UAS-Snail74b;UAS-RasV12The phenotype of the third-instar larva is that GFP is positioned at a head complex tissue (Cephalic complex), the larva is normal and not short and thick, after two cerebral hemispheres and abdominal ganglia (VNC) of the fruit fly are dissected, the tumor cells of the eye tissue marked by the GFP can be observed to migrate to the abdominal ganglia of the fruit fly, namely the drosophila melanogaster RasV12A Snail tumor migration model. At the same time, the user can select the desired position,eydrosophila strains of Flp, GFP, respectivelyw 1118 、UAS-RasV12AndUAS-Snail74bthe fruit fly strains are hybridized, and the genotypes of the selected later generations are respectivelyey-Flp, GFP/+、 ey-Flp, GFP /+;UAS-RasV12V. + andey-Flp, GFP/UAS-Snail74ball the third instar larvae had normal and non-short and thick body types with GFP in the complex tissues of the head (Cephalic complex). Wherein the content of the first and second substances,ey-Flp, GFP/+the drosophila melanogaster is a wild type control group,ey-Flp, GFP/+;UAS-RasV12the drosophila of/+ is a control group in which tumor cells only grow in situ and do not migrate,ey-Flp, GFP/UAS-Snail74bdrosophila of (a) is a control group over-expressing Snail alone neither induced tumor proliferation in situ nor caused migration.
(2) Collecting target drosophila melanogaster in filial generations, dissecting complex tissues of the heads and abdominal ganglia of drosophila melanogaster third-instar larvae, and taking pictures under a fluorescence microscope.
Brief introduction to model
FIGS. 5A-D are head complex tissue (Cephalic complex) anatomies of the third instar larvae stage of Drosophila melanogaster, where EA represents the eye-insect disc (eye-anti disc), BH represents the two cerebral hemispheres of Drosophila melanogaster, and VNC represents the Ventral ganglion (Ventral nerve cord, VNC).
Using FLP-FRT mediated mitotic recombination in conjunction with the Gal4/UAS system, we found sustained expression of the highly activated oncogene Ras (Ras) specifically in the ocular adult disc as compared to the normal group control (FIG. 5A, A') (Ras)V12) Can promote the in situ overgrowth of tumor cells (figure)5B, B'). On this basis, co-expression of the transcription factor Snail synergistically induced tumor growth and the tumor cells developed a metastatic phenotype towards the ventral ganglion (tumor cells were labeled with GFP green fluorescent protein, fig. 5C, C '), whereas expression of Snail alone did not (fig. 5D, D'). RasV12The Snail tumor migration model was thus successfully established. FIG. 5 shows that increasing Snail promotes tumor growth and RasV12Migration of the cells. Researchers can use the model to carry out large-scale genetic screening, search gene mutation or RNAi for inhibiting tumor migration, clarify the molecular mechanism for regulating tumor migration, and carry out functional verification on homologues thereof in mammalian cells so as to provide a new drug target and a new treatment scheme for clinical treatment of cancer.
Claims (8)
1. Drosophila melanogaster RasV12The method for establishing the Snail tumor migration model is characterized by comprising the following steps of: adopting specific drosophila melanogaster strain to carry out hybridization and then culturing and screening to obtain drosophila melanogaster RasV12A Snail tumor migration model; the method specifically comprises the following steps:
(1) will be provided withUAS-Snail74bThe drosophila strain is hybridized with Sp/Cyo and Sb/TM6B.Tb drosophila strain, and the genotype is selected from the progenyUAS-Snail74b/Sp;+Male fruit flies of Sb; simultaneously, Sp/Cyo, Sb/TM6B.Tb drosophila strains are combinedUAS-RasV12The fruit fly strains are hybridized, and the genotype is selected from the offspring+/Cyo;UAS-RasV12 /Tm6b.tb female drosophila; then will obtainUAS-Snail74b/Sp;+Male drosophila/Sb and +/Cyo, UAS-RasV12Female drosophila/TM6B.Tb is hybridized, and the genotype is selected from the progenyUAS-Snail74b/Cyo;UAS-RasV12Male fruit flies of Sb; then, will obtainUAS-Snail74b/Cyo;UAS-RasV12Hybridizing male Sb drosophila with female drosophila of Sb/SM6B-TM6B.Tb strain with genotype Sp and selecting genotype asUAS-Snail74b;UAS-RasV12the/SM 6B-TM6B.Tb drosophila melanogaster with proper male and female parts to form the Stock strain；
(2) Will be provided withey-Flpact>y+>Gal4UASGFP Drosophila strains and finally obtained in step (1)UAS-Snail74b;UAS-RasV12the/SM 6B-TM6B.Tb drosophila strain is hybridized, and the genotype is selected from the offspringey-Flpact>y+>Gal4UAS-GFP/UAS-Snail74b;UAS-RasV12The third instar larva with GFP phenotype and non-squash body type is drosophila melanogaster RasV12A Snail tumor migration model.
2. The drosophila melanogaster Ras of claim 1V12The method for establishing the Snail tumor migration model is characterized by comprising the following steps of: in the step (1)UAS-Snail74bThe drosophila strain and Sp/Cyo, Sb/TM6B.Tb the drosophila strain can be combined with parents in any way when the drosophila strain is hybridized; Sp/Cyo, Sb/TM6B.Tb Drosophila strain andUAS-RasV12the male and female parents can be combined randomly when the fruit fly strains are hybridized; in the step (2)ey-Flpact>y+>Gal4UASGFP Drosophila strains and finally obtained in step (1)UAS-Snail74b;UAS-RasV12The parents can be combined in any way when the/SM 6B-TM6B.Tb drosophila strain is hybridized.
3. The drosophila melanogaster Ras of claim 1V12The method for establishing the Snail tumor migration model is characterized by comprising the following steps of: the formula of the culture medium used in the culture process comprises 135g of brown sugar, 7g of agar, 85g of corn flour, 8g of yeast, 4ml of propionic acid and 1000ml of water.
4. The drosophila melanogaster Ras of claim 1V12The method for establishing the Snail tumor migration model is characterized by comprising the following steps of: the culture conditions are as follows: the temperature is constant at 20-30 ℃ and the humidity is 50% -60%.
5. The drosophila melanogaster Ras of claim 1V12The method for establishing the Snail tumor migration model is characterized by comprising the following steps of: step (1) and step (2)The process of crossing different drosophila strains is as follows: anaesthetizing fruit fly with CO2The operation of selecting male and female, hybridizing and observing phenotype is carried out on the flat plate; virgin flies which have not been mated must be collected before hybridization experiments are carried out; the method for selecting the virgins comprises the steps of completely removing the imagoes in the stock bottle, and collecting the female fruit flies which are just eclosion every 8 hours, namely the virgins.
6. The method of any one of claims 1 to 5 for constructing Drosophila melanogaster RasV12Application in Snail tumor migration model.
7. Drosophila melanogaster Ras constructed by the method of any one of claims 1 to 5V12A Snail tumor migration model.
8. The Drosophila melanogaster Ras of claim 4V12The method for establishing the Snail tumor migration model is characterized by comprising the following steps of: the culture conditions are as follows: the temperature is constant at 25 ℃ and the humidity is 50-60%.
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| CN113100178A (en) * | 2021-04-07 | 2021-07-13 | 华北理工大学 | Method for establishing drosophila melanogaster tumor invasion model |
| CN114258897B (en) * | 2022-01-05 | 2022-10-21 | 同济大学 | Method for establishing drosophila melanogaster model for inducing high-frequency transfer of colon cancer |
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