CN108184770A - A kind of Drosophila melanogaster RasV12;Snail tumours migrate the method for building up of model - Google Patents
A kind of Drosophila melanogaster RasV12;Snail tumours migrate the method for building up of model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The present invention discloses a kind of Drosophila melanogaster RasV12;Snail tumours migrate the method for building up of model, are cultivated after being hybridized using specific drosophila strains and screen acquisition Drosophila melanogaster RasV12;Snail tumours migrate model；The Drosophila melanogaster Ras of the method for the present invention structureV12;Snail tumours migrate model, researcher can carry out large-scale genetic screening using this model, find gene mutation or the RNAi of tumor migration inhibition, illustrate the molecular mechanism of its modulate tumor migration, and functional verification is carried out to its homologue in mammalian cell, to provide new drug target and therapeutic scheme for the clinical treatment of cancer.
Description
Technical field
The invention belongs to biogenetics technical fields, and in particular to a kind of Drosophila melanogaster RasV12;Snail tumours are migrated
The method for building up of model.
Background technology
Drosophila genetics operating system has Gal4/UAS double expressions system and FLP/FRT systems.Wherein,（One）Gal4/UAS is bis-
Expression system：Gal4/UAS systems are transgenic technology systems the most commonly used in drosophila, and foreign gene or RNAi can be allowed to exist
Specific cells or tissue selectivity expression.Galactolipin adjusts upstream promoter element（galactose-regulated
upstream promoter element 4）, abridge Gal4, is one that prokaryotes lactose operon is similar in yeast
Transcription activator.Upstream activating sequenceUAS（upstream activate sequense）, it is another similar high in yeast
The sequence of eucaryote enhancer.Gal4 by withUASIt is combined, adjusts the expression with galactose metabolism related gene.1993
Year, target gene X is connected to by scientistUASLater, it is established by transgenic technologyUAS- X drosophila strains, then with it is specific
Gal4 drosophila strains hybridization, can be obtained by while have in offspringY-Gal4 andUASThe drosophila of-X, so as to fulfill special
Property in Y tissue expression X genes（Bibliography 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）.Because drosophila gene group does not encode Gal4 transcription factors,
So the overexpression Gal4 in drosophila body, will not generate significant impact to the development of drosophila.Equally, it is inserted into drosophila bodyUASRegulating and controlling sequence will not have an impact drosophila.The scientist being established as by the use of drosophila as research model of this system
Advantageous, convenient, efficient genetic manipulation tool is provided in experimental design.Gal4/UAS double expressions system schematic such as Fig. 1
It is shown.
（Two）FLP/FRT systems：FLP is a kind of recombinase in yeast, it can identify the homologous target position of two 700bp
Point FRT (FLP recombination targets, FRTs), if two FRT segments are located at drosophila pair of homologous chromosome
Same loci, thermal shock induction FLP expression can mediate the two sites carry out mitosis recombination, generate recombination distal end it is pure
The daughter cell (Fig. 2) of conjunction（Bibliography 2, Zhang S. P. and Xue L., [Progress on cell lineage
analysis in Drosophila melanogaster]. Yi Chuan, 2012, 34: 819-28）.Research finds FRT
The mitosis recombination efficiency of mediation is far above other modes, and recombinating the site of generation can also artificially control.In addition, thermal shock pair
The injury of cell is also smaller.Therefore, researcher can probe into the cell of a small amount of gene alteration in wild type by FLP/FRT technologies
Growing state under cellular environment, so as to further study the molecular mechanism of cell competition.FLP/FRT systemic effects principle is illustrated
Figure is as shown in Figure 2.
Drosophila as research human diseases model organism, with mammal not only basic biology, physiology and
Nervous system function etc. is more similar, and drosophila has its unique advantage as model organism.Research table in recent years
Bright, drosophila and the mankind are very high in the conservative of tumour generation signal path etc., and drosophila has very strong science of heredity
Operability is one of effective model of oncology studies, occurs available for research human tumor, development, shifts equimolecular
Mechanism.Recent study personnel have been set up much being used for the transgenic drosophila model for studying particular cancers, but new particular cancers
The foundation of transgenic drosophila model play an important role to illustrating tumorigenic molecular mechanism, it will be the clinical treatment of more cancers
New drug target and therapeutic scheme are provided.Therefore, the transgenic drosophila model for establishing new specific tumors be those skilled in the art urgently
Technical problem to be solved.
Invention content
The purpose of the present invention is to provide a kind of Drosophila melanogaster RasV12;Snail tumours are migrated the method for building up of model and its are answered
With.
Another object of the present invention is to provide the Drosophila melanogaster Ras of above method foundationV12;Snail tumours migrate mould
Type.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of Drosophila melanogaster RasV12;Snail tumours migrate the method for building up of model, are hybridized using specific drosophila strains
After cultivate and screen and obtain Drosophila melanogaster RasV12;Snail tumours migrate model；Specifically include following steps：
（1）It willUAS-Snail74bDrosophila strains and Sp/Cyo;Sb/TM6B.Tb drosophila strains are hybridized, and are chosen in offspring
Genotype isUAS-Snail74b/Sp;+/ Sb Male Drosophilas；Simultaneously by Sp/Cyo;Sb/TM6B.Tb drosophila strains andUAS-
RasV12Drosophila strains are hybridized, and genotype is chosen in offspring and is+/Cyo;UAS-RasV12 /TM6B.Tb female Drosophilas；So
Afterwards by acquisitionUAS-Snail74b/Sp;+/ Sb Male Drosophilas and +/Cyo;UAS-RasV12/ TM6B.Tb female Drosophilas carry out miscellaneous
It hands over, genotype is chosen in offspring isUAS-Snail74b/Cyo;UAS-RasV12/ Sb Male Drosophilas；Next, it will obtainUAS-Snail74b/Cyo;UAS-RasV12/ Sb Male Drosophilas and genotype are Sp;The female of Sb/SM6B-TM6B.Tb strains
Drosophila is hybridized, and genotype is chosen in offspring and isUAS-Snail74b;UAS-RasV12/ SM6B-TM6B.Tb drosophilas, male and female
In right amount, Stock strains are built up；
（2）It willey-Flp act>y+>Gal4 UAS-GFP（It is abbreviated asey-Flp, GFP）Drosophila strains and step（1）In it is final
It obtainsUAS-Snail74b;UAS-RasV12/ SM6B-TM6B.Tb drosophila strains are hybridized, and genotype is chosen in offspring
Forey-Flp, GFP/UAS-Snail74b;UAS-RasV12/+third-instar larvae, GFP, build are non-short and thick to have for phenotype, as black
Abdomen drosophila RasV12;Snail tumours migrate model.
Step（1）InUAS-Snail74bDrosophila strains and Sp/Cyo;It is close when Sb/TM6B.Tb drosophila strains are hybridized
This male and female arbitrarily combines；Sp/Cyo;Sb/TM6B.Tb drosophila strains andUAS-RasV12It is close when drosophila strains are hybridized
This male and female arbitrarily combines；Step（2）Iney-Flp act>y+>Gal4 UAS- GFP drosophila strains and step（1）In it is final
It obtainsUAS-Snail74b;UAS-RasV12The arbitrarily combination of parent's male and female is equal when/SM6B-TM6B.Tb drosophila strains are hybridized
It can.
The formula of culture medium used in the incubation is brown sugar 135g, agar 7g, corn flour 85g, yeast 8g, third
Sour 4ml and water 1000ml.
The condition of the culture is：20 ~ 30 DEG C of constant temperature of temperature（Preferably 25 DEG C）, humidity 50%-60%.
It is by the process that drosophila strains hybridize：After drosophila is anaesthetized, it is being connected with CO2Tablet on select it is female
Male, hybridization and the operation of observation phenotype；The virgin fly not mated must be collected before doing hybrid experiment；The virgin fly selection side
Method is all to remove the adult in original seeds bottle, and it is virgin fly that the female drosophila just to have sprouted wings was hereafter collected every 8 hours.
Above-mentioned method is in structure Drosophila melanogaster RasV12;Snail tumours migrate the application in model.
The Drosophila melanogaster Ras of above-mentioned method structureV12;Snail tumours migrate model.
Beneficial effects of the present invention：
The Drosophila melanogaster Ras of the method for the present invention structureV12;Snail tumours migrate model, and researcher can be carried out big using this model
The genetic screening of scale finds gene mutation or the RNAi of tumor migration inhibition, illustrates the molecular mechanism of its modulate tumor migration,
And functional verification is carried out to its homologue in mammalian cell, to provide new drug target for the clinical treatment of cancer
And therapeutic scheme.
Description of the drawings
Fig. 1 is Gal4/UAS double expression system schematics.
Fig. 2 is FLP/FRT systemic effect principle schematics.
Fig. 3 distinguishes schematic diagram for drosophila male and female.
Fig. 4 is drosophila hybrid flow diagram.
Fig. 5 promotes tumour growth and Ras for up-regulation SnailV12Cell is migrated.
Wherein figure A-D is the head complex organization of drosophila third-instar larvae phase（Cephalic complex）Internal anatomy, wherein,
EA represents eye imaginal discs（eye-antennal disc）, BH represents two cerebral hemispheres of drosophila, and VNC represents abdominal nerve section
(Ventral nerve cord, VNC）.
Specific embodiment
Embodiment 1 establishes Drosophila melanogaster RasV12;Snail tumours migrate model
First, the raising of drosophila and experiment condition
（One）The preparation of Drosophila medium
The drosophila strains and hybrid experiment of experiment raise drosophila in brown sugar-corn flour-yeast culture medium of standard,
The formula of middle culture medium is as follows：Brown sugar 135g, agar 7g, corn flour 85g, yeast 8g, propionic acid 4ml and water 1000ml.
Process for preparation：（1）Load weighted brown sugar and agar are poured into electric cooker together, suitable quantity of water is added in, is sufficiently stirred；
（2）It is heated to boiling；（3）It will be poured slowly into pot with the corn flour that water has fully dissolved, it is lasting to stir；（4）It is heated to boiling
It rises；（5）Object to be mixed is cooled to 80 DEG C or so, adds in the yeast dissolved in advance with warm water, is sufficiently stirred；（6）It adds in appropriate
Propionic acid solution is sufficiently stirred；（7）Culture medium is sub-packed in the glass tube of sterilizing；（8）It is In Shade to deposit beyond the Great Wall after cotton
It puts.
（Two）Experiment condition
25 DEG C of constant temperature of temperature, humidity 50%-60%, general raising is in the incubator of 25 DEG C of constant temperature and humidities or drosophila room.
（Three）The discriminating of drosophila male and female
（1）Build：Female drosophila build is larger, and male is smaller.（2）Abdomen end：Female drosophila abdomen ellipsoidal ends are slightly sharp, male drosophila
End blunt circle.（3）Belly back side：Female drosophila has apparent 5 blackstreaks, and male drosophila has 3, first 2 thin, latter 1 wide, until
The outside of belly, the visible abdomen end of naked eyes have an apparent stain.（4）Venter：Female drosophila has obvious 6 sternites, male drosophila
There are 4 sternites.（5）Sex comb：Topmost attached sufficient front-end surface in male drosophila first segment vola side has black mane-sex comb.（6）It hands over
Cercoids：Judge the most important difference of female male drosophila.Drosophila male and female distinguish that schematic diagram is as shown in Figure 3.
（Four）The anesthesia and hybridization of drosophila
The anesthesia of drosophila is carbon dioxide (CO2) gas anesthesia method.Parent drosophila as hybrid experiment, it is impossible to excessively
Otherwise anesthesia can influence the viability of drosophila.After the narcosis of drosophila and anesthesia dead difference using wing whether abduction as
Foundation.Narcose two wings of drosophila are still overlapped on back of the body abdomen, and dead drosophila wing leaves abdomen in abduction shape
State.
Drosophila is placed in and is connected with CO2Tablet on, drosophila can be anaesthetized within 3-5 seconds.After drosophila is anaesthetized, according to female drosophila and
The difference of male drosophila exophenotype collects the Male Drosophila of the female virgin fly not mated and health, according to hybrid experiment respectively
Flow will hybridize the parent of needs（Female fly and male fly）After being combined, it is placed in same drosophila pipe（Fresh culture is added
Base）Offspring is collected in raising, and observes the phenotype of hybridization target offspring.
Due to there is seminal vesicle in female drosophila reproductive organs, one time post-coitum can store a large amount of sperms, for fertilization of repeatedly ovulating
With, therefore the virgin fly not mated must be collected before doing hybrid experiment, otherwise experimental result is insecure.Choosing method：
Adult in original seeds bottle is all removed, the female drosophila just to have sprouted wings was hereafter collected every 8 hours, is put into spare in culture bottle.By
In the drosophila just to have sprouted wings, body is elongated and young nearly transparently tender, penetrates the shell of chitin from the outside of belly of abdomen, can see
To intraperitoneal black alimentary canal.Therefore, it is virgin fly that the gastral female individuals of black, which can be observed,.
2nd, Drosophila melanogaster RasV12;Snail tumours migrate the foundation of model
1st, drosophila strains needed for model are established
（1）ey-Flp act>y+>Gal4 UAS- GFP, on No. second chromosome of drosophila, it is impossible to homozygous, specific genotype
For：w 1118 ;ey-Flp act>y+>Gal4 UAS- GFP/Cyo is ordered in Chinese Academy of Sciences's Shanghai Sheng Ke institutes biochemistry and cell institute drosophila
Resource and technology platform.
（2）UAS-RasV12, it, can be homozygous on drosophila third chromosome, specific genotype is：w;UAS-
RasV12, order in the Bloomington of Indiana universities of the U.S.DrosophilaStock Center, number：
BL64159。
（3）UAS-Snail74b, it is wild type Snail, on No. second chromosome of drosophila, which is
Yellow, can be homozygous, and specific genotype is：w 1118 ;UAS-Snail74b, order in Shanghai Sheng Ke institutes of Chinese Academy of Sciences biochemistry and cell
Institute's drosophila resource and technology platform.
（4）Sp/Cyo;Sb/TM6B.Tb, the tool drosophila of No. two No. three chromosomes of drosophila are ordered in Chinese Academy of Sciences's Shanghai life
Institute of section biochemistry and cell institute drosophila resource and technology platform.
（5）Sp;Sb/SM6B-TM6B.Tb, the tool drosophila that No. two No. three chromosomes of drosophila are connected, is ordered in the Chinese Academy of Sciences
Shanghai Sheng Ke institutes biochemistry and cell institute drosophila resource and technology platform.
（6）w 1118 , on drosophila X chromosome, order in Tsinghua University drosophila center, number：THJ0265.
, result genotype it is as follows
Fig. 5 A：ey-Flp act>y+>Gal4 UAS-GFP/+
Fig. 5 B：ey-Flp act>y+>Gal4 UAS-GFP/+;UAS-RasV12/+
Fig. 5 C：ey-Flp act>y+>Gal4 UAS-GFP/UAS-Snail74b;UAS-RasV12/+
Fig. 5 D：ey-Flp act>y+>Gal4 UAS-GFP/UAS-Snail74b
3rd, experimental method
（1）The female virgin fly gathered and healthy Male Drosophila are hybridized according to hybridization flow, drosophila hybrid flow is shown in
Fig. 4, detailed step are as follows：
A. willUAS-Snail74bDrosophila strains and Sp/Cyo;Sb/TM6B.Tb drosophila strains are hybridized, and are chosen in offspring
Genotype isUAS-Snail74b/Sp;+/ Sb Male Drosophilas；Simultaneously by Sp/Cyo;Sb/TM6B.Tb drosophila strains andUAS-
RasV12Drosophila strains are hybridized, and genotype is chosen in offspring and is+/Cyo;UAS-RasV12 /TM6B.Tb female Drosophilas；So
Afterwards by acquisitionUAS-Snail74b/Sp;+/ Sb Male Drosophilas and +/Cyo;UAS-RasV12/ TM6B.Tb female Drosophilas carry out miscellaneous
It hands over, genotype is chosen in offspring isUAS-Snail74b/Cyo;UAS-RasV12/ Sb Male Drosophilas；Next, it will obtainUAS-Snail74b/Cyo;UAS-RasV12/ Sb Male Drosophilas and genotype are Sp;The female of Sb/SM6B-TM6B.Tb strains
Drosophila is hybridized, and genotype is chosen in offspring and isUAS-Snail74b;UAS-RasV12The drosophila of/SM6B-TM6B.Tb, it is female
It is male appropriate, build up the drosophila strains that can pass on preservation.
B. willey-Flp act>y+>Gal4 UAS-GFP（It is abbreviated asey-Flp, GFP）In drosophila strains and step a most
It obtains eventuallyUAS-Snail74b;UAS-RasV12/ SM6B-TM6B.Tb drosophila strains are hybridized, and genotype is chosen in offspring
Forey-Flp, GFP/UAS-Snail74b;UAS-RasV12/+third-instar larvae, phenotype be head complex organization（Cephalic
complex）Place has GFP, build normal non-short and thick, dissects two cerebral hemispheres of drosophila and abdominal nerve section (Ventral
Nerve cord, VNC）Afterwards, it is observed that the tumour cell of the ocular tissue of GFP labels has migrated the abdominal nerve of drosophila
Section, as Drosophila melanogaster RasV12;Snail tumours migrate model.MeanwhileeyThe drosophila strains of-Flp, GFP respectively withw 1118 、UAS-RasV12WithUAS-Snail74bDrosophila strains are hybridized, and genotype is chosen in offspring and is respectivelyey-Flp, GFP/+、 ey-Flp, GFP /+;UAS-RasV12/+andey-Flp, GFP/UAS-Snail74bThird-instar larvae, phenotype is head
Complex organization（Cephalic complex）Place has GFP, the build normal non-short and thick.Wherein,ey-Flp, GFP/+Drosophila for open country
Raw type control group,ey-Flp, GFP/+;UAS-RasV12/+drosophila for the nonmigratory control group of tumour cell growth in situ,ey-Flp, GFP/UAS-Snail74bDrosophila moved to be individually overexpressed Snail neither induced tumor Proliferations nor cause
The control group of shifting.
（2）Collect the target drosophila in filial generation, to drosophila third-instar larvae head complex organization and abdominal nerve section into
Row dissection is placed in taking pictures under fluorescence microscope.
, model brief introduction
Fig. 5 A-D are the head complex organization of drosophila third-instar larvae phase（Cephalic complex）Internal anatomy, wherein EA represent eye
Imaginal discs（eye-antennal discs）, BH represents two cerebral hemispheres of drosophila, and VNC represents abdominal nerve section (Ventral
Nerve cord, VNC）.
It is recombinated using the FLP-FRT mitosis mediated and combines Gal4/UAS systems, compared with normal group control（Figure
5A, A '）, it has been found that the specifically overactive oncogene Ras of continuous expression in eye imaginal discs（RasV12）, can promote
Into the undue growth in situ of tumour cell（Fig. 5 B, B '）.On this basis, when co-expressing transcription factor Snail, it can cooperate with and enough lure
Tumour growth is led, and phenotype is migrated in the meeting ventrad neuromere generation of these tumour cells（Tumour cell GFP green fluorescences
Protein labeling, Fig. 5 C, C '）, and single expression Snail does not have then and migrates phenotype（Fig. 5 D, D '）.RasV12;Snail tumours are moved
Therefore model of moving is successfully established.Fig. 5 promotes tumour growth and Ras for up-regulation SnailV12Cell is migrated.Researcher can utilize this
Model carries out large-scale genetic screening, finds gene mutation or the RNAi of tumor migration inhibition, illustrates the migration of its modulate tumor
Molecular mechanism, and in mammalian cell to its homologue carry out functional verification, provided to the clinical treatment for cancer
New drug target and therapeutic scheme.
Claims (7)
1. a kind of Drosophila melanogaster RasV12;Snail tumours migrate the method for building up of model, it is characterised in that：Using specific drosophila
Strain, which is cultivated and screened after being hybridized, obtains Drosophila melanogaster RasV12;Snail tumours migrate model；Specifically include following steps：
（1）It willUAS-Snail74bDrosophila strains and Sp/Cyo;Sb/TM6B.Tb drosophila strains are hybridized, and are chosen in offspring
Genotype isUAS-Snail74b/Sp;+/ Sb Male Drosophilas；Simultaneously by Sp/Cyo;Sb/TM6B.Tb drosophila strains andUAS-
RasV12Drosophila strains are hybridized, and genotype is chosen in offspring and is+/Cyo;UAS-RasV12 /TM6B.Tb female Drosophilas；So
Afterwards by acquisitionUAS-Snail74b/Sp;+/ Sb Male Drosophilas and +/Cyo;UAS-RasV12/ TM6B.Tb female Drosophilas carry out miscellaneous
It hands over, genotype is chosen in offspring isUAS-Snail74b/Cyo;UAS-RasV12/ Sb Male Drosophilas；Next, it will obtainUAS-Snail74b/Cyo;UAS-RasV12/ Sb Male Drosophilas and genotype are Sp;The female of Sb/SM6B-TM6B.Tb strains
Drosophila is hybridized, and genotype is chosen in offspring and isUAS-Snail74b;UAS-RasV12/ SM6B-TM6B.Tb drosophilas, male and female
In right amount, Stock strains are built up；
（2）It willey-Flp act>y+>Gal4 UAS-GFP（It is abbreviated asey-Flp, GFP）Drosophila strains and step（1）In it is final
It obtainsUAS-Snail74b;UAS-RasV12/ SM6B-TM6B.Tb drosophila strains are hybridized, and genotype is chosen in offspring
Forey-Flp, GFP/UAS-Snail74b;UAS-RasV12/+third-instar larvae, GFP, build are non-short and thick to have for phenotype, as black
Abdomen drosophila RasV12;Snail tumours migrate model.
2. Drosophila melanogaster Ras according to claim 1V12;Snail tumours migrate the method for building up of model, and feature exists
In：Step（1）InUAS-Snail74bDrosophila strains and Sp/Cyo;Parent's male and female when Sb/TM6B.Tb drosophila strains are hybridized
Arbitrary combination；Sp/Cyo;Sb/TM6B.Tb drosophila strains andUAS-RasV12Parent's male and female when drosophila strains are hybridized
Arbitrary combination；Step（2）Iney-Flp act>y+>Gal4 UAS- GFP drosophila strains and step（1）In finally obtainUAS-Snail74b;UAS-RasV12Parent's male and female arbitrarily combine when/SM6B-TM6B.Tb drosophila strains are hybridized.
3. Drosophila melanogaster Ras according to claim 1V12;Snail tumours migrate the method for building up of model, and feature exists
In：The formula of culture medium used in the incubation is brown sugar 135g, agar 7g, corn flour 85g, yeast 8g, propionic acid 4ml
With water 1000ml.
4. Drosophila melanogaster Ras according to claim 1V12;Snail tumours migrate the method for building up of model, and feature exists
In：The condition of the culture is：20 ~ 30 DEG C of constant temperature of temperature, humidity 50%-60%；Preferably 25 DEG C of constant temperature of temperature, humidity 50%-
60%。
5. Drosophila melanogaster Ras according to claim 1V12;Snail tumours migrate the method for building up of model, and feature exists
In：Step（1）And step（2）It is by the process that different drosophila strains hybridize：After drosophila is anaesthetized, it is being connected with CO2It is flat
It carries out selecting male and female, hybridization and the operation for observing phenotype on plate；The virgin fly not mated must be collected before doing hybrid experiment；Institute
It is all to remove the adult in original seeds bottle to state virgin fly choosing method, and the female drosophila just to have sprouted wings was hereafter collected every 8 hours i.e.
For virgin fly.
6. any method is in structure Drosophila melanogaster Ras in claim 1 ~ 5V12;Snail tumours migrate answering in model
With.
7. the Drosophila melanogaster Ras of any method structure in claim 1 ~ 5V12;Snail tumours migrate model.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109601487A (en) * | 2018-12-26 | 2019-04-12 | 同济大学 | A kind of Drosophila melanogaster tumor model and its application |
| CN112956453A (en) * | 2021-04-07 | 2021-06-15 | 华北理工大学 | Method for establishing drosophila melanogaster insulin antidiabetic model |
| CN113100178A (en) * | 2021-04-07 | 2021-07-13 | 华北理工大学 | Method for establishing drosophila melanogaster tumor invasion model |
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| CN114342883A (en) * | 2022-01-21 | 2022-04-15 | 西湖大学 | Construction method and application of drosophila organoorgan communication dual-system tumor screening model |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050005311A1 (en) * | 2003-04-28 | 2005-01-06 | Liotta Lance A. | High throughput screening for cancer genes |
| CN103238565A (en) * | 2012-12-13 | 2013-08-14 | 国家海洋局第三海洋研究所 | Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model |
| CN103865933A (en) * | 2013-11-30 | 2014-06-18 | 济南大学 | Application of WAP (whey acidic protein) gene in transgenosis of fruit fly |
| CN105283552A (en) * | 2013-03-13 | 2016-01-27 | 澳大利亚核科学和技术组织 | Transgenic non-human organisms with non-functional TSPO genes |
| CN105636434A (en) * | 2013-08-20 | 2016-06-01 | 托斯克公司 | Non-mammalian RAS transgenic animal model |
| CN106259222A (en) * | 2016-08-29 | 2017-01-04 | 中国科学院上海有机化学研究所 | A kind of method of the living Animal Models built for studying neuron autophagy |
-
2018
- 2018-01-19 CN CN201810055291.2A patent/CN108184770B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050005311A1 (en) * | 2003-04-28 | 2005-01-06 | Liotta Lance A. | High throughput screening for cancer genes |
| CN103238565A (en) * | 2012-12-13 | 2013-08-14 | 国家海洋局第三海洋研究所 | Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model |
| CN105283552A (en) * | 2013-03-13 | 2016-01-27 | 澳大利亚核科学和技术组织 | Transgenic non-human organisms with non-functional TSPO genes |
| CN105636434A (en) * | 2013-08-20 | 2016-06-01 | 托斯克公司 | Non-mammalian RAS transgenic animal model |
| CN103865933A (en) * | 2013-11-30 | 2014-06-18 | 济南大学 | Application of WAP (whey acidic protein) gene in transgenosis of fruit fly |
| CN106259222A (en) * | 2016-08-29 | 2017-01-04 | 中国科学院上海有机化学研究所 | A kind of method of the living Animal Models built for studying neuron autophagy |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109601487A (en) * | 2018-12-26 | 2019-04-12 | 同济大学 | A kind of Drosophila melanogaster tumor model and its application |
| CN109601487B (en) * | 2018-12-26 | 2020-07-14 | 同济大学 | Drosophila melanogaster tumor model and application thereof |
| CN112956453A (en) * | 2021-04-07 | 2021-06-15 | 华北理工大学 | Method for establishing drosophila melanogaster insulin antidiabetic model |
| CN113100178A (en) * | 2021-04-07 | 2021-07-13 | 华北理工大学 | Method for establishing drosophila melanogaster tumor invasion model |
| CN114258897A (en) * | 2022-01-05 | 2022-04-01 | 同济大学 | Method for establishing drosophila melanogaster model for inducing high-frequency transfer of colon cancer |
| CN114342883A (en) * | 2022-01-21 | 2022-04-15 | 西湖大学 | Construction method and application of drosophila organoorgan communication dual-system tumor screening model |
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