CN107227352A - The detection method of GPR120 gene expressions based on eGFP and application - Google Patents

The detection method of GPR120 gene expressions based on eGFP and application Download PDF

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CN107227352A
CN107227352A CN201710443881.8A CN201710443881A CN107227352A CN 107227352 A CN107227352 A CN 107227352A CN 201710443881 A CN201710443881 A CN 201710443881A CN 107227352 A CN107227352 A CN 107227352A
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egfp
gpr120
gene expression
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赵玉峰
苏兴利
徐曦
闫爱丽
谢荣
魏兰兰
刘英光
陈镝
梁向艳
赵妍妍
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Xian Medical University
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Abstract

EGFP fragments fixed point, is inserted at the terminator codon TAA of GPR120 genes by the detection method of the GPR120 gene expressions disclosed by the invention based on eGFP by CRISPR/Cas9 technologies, obtains the transgenic models mouse that eGFP marks GPR120 positive cells；Then application fluorescence analyser is excited is measured to the positive cell fluorescence intensity of mouse.The present invention by monitoring GPR120 gene expressions in real time, error between control and reduction group, ensure result reliability, make up and overcome existing detection method to compare and produce between different tissues cell group variability, can not be the problem of the change of living cells level monitoring gene expression；The present invention determines GPR120 gene expression doses at once after cell is collected, and eliminates operation and the courses of reaction such as RNA extractions and reverse transcription, PCR, makes GPR120 gene expression detections easier.

Description

The detection method of GPR120 gene expressions based on eGFP and application
Technical field
The invention belongs to eGFP biologic applications technical fields, and in particular to a kind of GPR120 gene expressions based on eGFP Detection method, the invention further relates to a kind of application based on eGFP in GPR120 gene expression detections.
Background technology
GPR120(G-protein coupled receptor 120；Also known as free fatty acid receptor 4, FFAR4 it is) fatty acid receptors family a member, belongs to g protein coupled receptor (GPCR), can be by long-chain fat acid active, especially with n-3 Unrighted acid activation capability is most strong.GPR120 is main in many tissue expressions such as brain, hypophysis, lung, tongue, stomach and intestine, adipose tissue It is distributed in a variety of endocrine cells of stomach and intestine, fat cell, macrophage, skeletonization and osteoclast and taste buds cell. GPR120 activation can stimulate the gastrointestinal hormones such as GLP-1, CCK, GIP to secrete, and influence the endocrine metabolism activity of body, with obesity etc. Metabolic disorder is closely related.
GPR120 expressions height significantly affects its cell adjustment effect, and the research of GPR120 expression regulations will promotion pair The understanding of GPR120 functions and the Metabolism regulation target molecule having found that it is likely that, for GPR120 target drugs research and development provide it is certain according to According to.Gene expression research institute common technology means have inverse transcription polymerase chain reaction (RT-PCR) and Northern traces at present Hybridize (Northern Blot), although both approaches technology maturation, be widely used in the detection of gene expression dose, There is also certain shortcoming：One is that experimentation is relatively complicated, two be can not living cells level monitoring gene expression change.
The content of the invention
It is an object of the invention to provide a kind of detection method of GPR120 gene expression doses based on eGFP and its application, Solve experimental procedure in the detection of existing GPR120 gene expression doses it is cumbersome, can not be in living cells level monitoring gene expression Change the problem of, the detection for GPR120 gene expression doses provides new approaches.
The technical solution adopted in the present invention is, applications of the eGFP in the detection of GPR120 gene expression doses.
It is a feature of the present invention that
EGFP and GPR120 gene expression doses it is linear, i.e., with the increase of eGFP fluorescence average intensity values, The increase of GPR120 gene expression doses.
Another technical scheme of the present invention is, the detection side of the GPR120 gene expression doses based on eGFP Method：
Step 1, eGFP fragments are pinpointed to the termination codon for being inserted into GPR120 genes by CRISPR/Cas9 technologies first At sub- TAA, eGFP is expressed and is expressed with GPR120, obtain the transgenic models mouse that eGFP marks GPR120 positive cells；
Step 2, then positive cell fluorescence intensity of the application fluorescence analyser to mouse under 488nm laser excitations is carried out Determine.
It is a feature of the present invention that
The fluorescence intensity of step 2 is expressed by unicellular average fluorescent strength.
The specific steps of step 2：
Step 2.1, the laser intensity index for carrying out fluorescence analyser using the mouse cell do not demarcated by eGFP is corrected；
Step 2.2, in take-up step 1 different tissues of transgenic models mouse eGFP positive cells, by through step Fluorescence analyser after 2.1 corrections carries out fluorescent strength determining, obtains the relation between eGFP and GPR120 gene expression doses； The gene expression dose of GPR120 in eGFP positive cells, checking eGFP and GPR120 genes are detected by RT-PCR method simultaneously Relation between expression.
Present invention has the advantages that：
A) present invention accomplishes error in itself cross-reference, control group by monitoring GPR120 gene expressions in real time, reduces Error between group, it is ensured that measurement result is more reliable, so as to make up and overcome the methods such as RT-PCR and Northern Blot to not With the variability for comparing and producing between histocyte group, can not asking in the change of living cells level monitoring gene expression Topic；
B) present invention is collected determines GPR120 gene expression doses at once after cell, eliminate RNA extractions, RNA reverse transcriptions With operation and the course of reaction such as PCR, make GPR120 gene expression detections more simple and efficient.
Brief description of the drawings
Fig. 1 be GPR120-ires-eGFP transgenic mices histocyte in eGFP fluorescencepositive cells, wherein, Figure 1A For GPR120-ires-eGFP transgenic mouse tissues under 488nm laser excitations eGFP fluorescencepositive cells figure, Figure 1B for figure The common light microscopic figure of 1A tissue, Fig. 1 C are that wild-type mice is organized in cytological map under 488nm laser excitations, and Fig. 1 D are Fig. 1 C's The common light microscopic figure of tissue；
Fig. 2 is the amplification curve diagram in mouse tissue, wherein, Fig. 2A is the group of GPR120-ires-eGFP transgenic mices The middle GPR120 small amplification curves of RT-PCR are knitted, Fig. 2 B are eGFP in the tissue of GPR120-ires-eGFP transgenic mices The small amplification curves of RT-PCR, the small amplification curves of RT-PCR that Fig. 2 C are GPR120 in wild-type mice tissue, Fig. 2 D are wild type The small amplification curves of eGFP RT-PCR in mouse tissue；
Fig. 3 is eGFP fluorescence intensities and GPR120 genes in GPR120-ires-eGFP transgenic mice different tissues cells Relation between expression；
Fig. 4 is that eGFP is glimmering after GPR120-ires-eGFP transgenic mice peritoneal macrophages are handled through lipopolysaccharides in vitro Graph of a relation between luminous intensity and GPR120 gene expression doses, wherein, Fig. 4 A are GPR120 gene expression doses in control group With the graph of a relation through lipopolysaccharides treatment group, Fig. 4 B are eGFP mean fluorescence intensities in control group and relation through lipopolysaccharides treatment group Figure, Fig. 4 C are the graphs of a relation of GPR120 gene expression doses and cell eGFP mean fluorescence intensities.
Embodiment
The present invention is described in detail with reference to the accompanying drawings and detailed description.
The present invention is determined using the model mice of eGFP mark GPR120 positive cells as object using fluorescence microscopy The fluorescence intensity of mouse eGFP positive cells, correlation analysis is carried out with the GPR120 gene expression doses of eGFP positive cells, The linear relationship between the fluorescence intensity of eGFP positive cells and GPR120 gene expression doses is determined, makees eGFP fluorescence intensities Foundation is provided for a reliable Testing index of GPR120 gene expression doses.
The detection method of GPR120 gene expression doses based on eGFP：
Step 1, eGFP fragments are pinpointed to the termination codon for being inserted into GPR120 genes by CRISPR/Cas9 technologies first At sub- TAA, eGFP is expressed and is expressed with GPR120, obtain the transgenic models mouse that eGFP marks GPR120 positive cells, So eGFP is expressed and expressed with GPR120, and is separated from each other in mRNA level in-site, eGFP and GPR120, it is ensured that GPR120 eggs White function and metabolic process is unaffected；
Step 2, then positive cell fluorescence intensity of the application fluorescence analyser to mouse under 488nm laser excitations is carried out Determine, and expressed by unicellular average fluorescent strength,
Step 2.1, the laser intensity index for carrying out fluorescence analyser using the mouse cell do not demarcated by eGFP is corrected；
Step 2.2, in take-up step 1 different tissues of transgenic models mouse eGFP positive cells, by through step Fluorescence analyser after 2.1 corrections carries out fluorescent strength determining, obtains the relation between eGFP and GPR120 gene expression doses； The gene expression dose of GPR120 in eGFP positive cells, checking eGFP and GPR120 genes are detected by RT-PCR method simultaneously Relation between expression.
1. experimental subjects
The eGFP of preparation, which is marked in GPR120 model mice, cell tissue, can see eGFP positive cells, in 488nm laser Excite down and send green fluorescence, it is specific as shown in Figure 1：Figure 1A is positive for the fluorescence of GPR120-ires-eGFP transgenic mouse tissues Property cytological map, it is seen that positive cell, Figure 1B for Figure 1A tissue common light microscopic figure, Fig. 1 C be wild-type mice tissue cell Figure, has no fluorescencepositive cell, and Fig. 1 D are the common light microscopic figure of Fig. 1 C tissue.
2. intracellular rna is extracted and reverse transcription
GPR120 model mice and wild-type mice are marked in eGFP, gastric mucosa, the duodenum of mouse are extracted respectively The RNA of the histoorgans such as mucous membrane, jejunal mucous membrane, mucous membrane of colon, adipose tissue, hypophysis, lungs is extracted and is carried out reverse transcription, specifically It is as follows：Each 1-2mg of above-mentioned tissue is taken, 350 μ L lysate RL are added per sample, tissue is ruptured using tissue disruption instrument, then will be all Solution is transferred in Filter column CS with 12000rpm centrifugation 2min, collects filtrate；350 μ L 70% are added into filtrate again Ethanol solution, be transferred to after mixing in adsorption column CR3 with 12000rpm centrifugation 1min, outwell the waste liquid in collecting pipe, will Adsorption column CR3 is put back in collecting pipe；350 μ L protein liquid removal RW1 are added into adsorption column CR3, with 12000rpm centrifugations 1min, outwells the waste liquid in collecting pipe, and adsorption column CR3 is put back in collecting pipe；80 μ L DNaseI are added into adsorption column CR3 Working solution, room temperature places 15min；350 μ L protein liquid removal RW1 are added into adsorption column CR3, with 12000rpm centrifugations 1min, outwells the waste liquid in collecting pipe, and adsorption column CR3 is put back in collecting pipe；500 μ L rinsing liquids are added into adsorption column CR3 RW, is stored at room temperature 2min, with 12000rpm centrifugation 1min, outwells the waste liquid in collecting pipe, adsorption column CR3 is put back into collection Guan Zhong；Repetition adds 500 μ L rinsing liquid RW into adsorption column CR3, is stored at room temperature 2min, with 12000rpm centrifugation 2min, The waste liquid in collecting pipe is outwelled, adsorption column CR3 is placed in room temperature placement 5min thoroughly to dry remnants rinsing liquid；By adsorption column CR3 is transferred in RNase-Free centrifuge tubes, adds 50 μ L Rnase-Free ddH2O, and room temperature places 2min, with 12000rpm speed Degree centrifugation 2min, obtains RNA solution.
ELIASA detects RNA concentration and purity, agarose gel electrophoresis detection RNA mass.2 are sequentially added in eight connecting legs μ L 5x gDNA Eraser Buffer, then 1 μ L gDNA Eraser are separately added into, then 1 μ g Total RNA are separately added into, most 10 μ L are complemented to RNase Free dH2O afterwards, are mixed, 5min is reacted at room temperature.Following reagent, 4 μ L are separately added into each pipe 5x PrimeScript Buffer2,4 μ LRNase Free dH2O, 1 μ L PrimeScript RT Enzyme Mix I and 1 μ L RT Primer Mix, overall reaction system is 20 μ L, is mixed.Reaction condition is 37 DEG C, 15min, 85 DEG C, 5s.After the completion of reaction By cDNA be stored in 4 DEG C it is standby, it is long-term to be transferred to -20 DEG C when preserving.
3.GPR120 the detection with eGFP gene expression doses
GPR120 and eGFP gene expression are detected using quantifying PCR method.6 μ L are sequentially added in eight connecting legs DH2O, 2 μ L templates cDNA, 2 μ L FAS primers, 10 μ L SYBR PremixEx TaqII (2x), overall reaction system is 20 μ L, is mixed It is even.
Reaction condition is：The first step is 94 DEG C, 5min, and second step is 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min circulations Carry out 40 times, the 3rd step is heating dissolving amplified production, obtains solubility curve.
Wherein, mouse GPR120 primer sequences are in overall reaction system：
Sense primer：5’-GTG CCG GGA CTG GTC ATT GTG-3’；
Anti-sense primer：5’-TTG TTG GGA CAC TCG GAT CTG G-3’.
EGFP primer sequences are in overall reaction system：
Sense primer：5’-TCT TCT TCA AGG ACG ACG GCA ACT-3’；
Anti-sense primer：5’-CCT TGA TGC CGT TCT TCT GCT TGT-3’.
Using beta-actin gene expressions as internal reference, beta-actin primer sequences are in overall reaction system：
Sense primer：5’-CGT TGG CAT CCA CGA AAC TA-3’；
Anti-sense primer：5’-GGT GCT GGG AGG TAC AGG G-3’.
Amplification curve as shown in Figure 2 is obtained to the analysis of experimental result expression.As Fig. 2A represents transgenic mice The small amplification curves of GPR120 RT-PCR in tissue, such as Fig. 2 B represent the small amplifications of RT-PCR of eGFP in the tissue of transgenic mice Curve, both of which is raised with the increase of amplification cycles number of times in certain phase.As Fig. 2 C represent wild-type mice tissue The middle GPR120 small amplification curves of RT-PCR, both of which is raised with the increase of amplification cycles number of times in certain phase, such as Fig. 2 D represent the small amplification curves of the RT-PCR of eGFP in wild-type mice tissue, and Dependence Results are without expression.
Using beta-actin gene expressions as internal reference, the GPR120 gene expression doses in each different tissues are carried out Analysis, and the green fluorescence intensity of the cells marked of eGFP in each tissue is measured using fluorescence microscope.As a result table It is bright, in tissue GPR120 expressions and organize Green fluorecyte fluorescence intensity between be in notable positive correlation, as a result such as Shown in Fig. 3.
Embodiment
The pulmonary alveolar macrophage of the GPR120 of eGFP marks model mice is cultivated, specific method is as follows：In vitro culture is adopted The model mice that eGFP marks GPR120 is put to death with cervical dislocation method, carried out disinfection processing with 75% chronic ethanol treated mice；With 10ml syringes draw PBS 6ml or so, remove bubble standby；Neck is cut off with eye scissors to thoracic cavity exocuticle, separation Pars cervicalis tracheae threading is standby, and cutting off thoracic cavity exposes lung, and tracheae front end is clamped with tweezers, cuts short tracheae and inserts syringe needle point Enter tracheae and tightened with line；Being slowly injected into PBS 2-3ml is full of lung, suctions out intrapulmonary solution and is slowly injected into again, circulates 3-5 times, intrapulmonary PBS injection centrifuge tube is drawn, gained is the solution containing pulmonary macrophage；By centrifuge tube trim in low temperature Horizontal centrifuge centrifuges 6min, and rotating speed is 1200rpm；Supernatant is sucked, substrate is pulmonary macrophage.
Cell culture fluid is added into pulmonary macrophage, is fully mixed, is planted in culture dish, make every group of culture dish inner cell number Uniformly, date and title are put on, puts in 37 DEG C of CO2 incubators and cultivates.After culture 8 hours, cell packet, one group of addition 200ng/ml lipopolysaccharides (LPS) is handled 24 hours, and another set is control group.
Then, the fluorescence intensity of the cells marked of eGFP in each tissue is measured using fluorescence microscope, measured After fluorescence intensity, the RNA of cell is collected, GPR120 gene expression doses are observed using quantifying PCR method after reverse transcription.Knot Fruit shows that as shown in Figure 4 A, the GPR120 gene expressions of pulmonary alveolar macrophage are compared with LPS treatment groups, and control group is significantly raised； As shown in Figure 4 B, while the eGFP mean fluorescence intensities of pulmonary alveolar macrophage are compared with LPS treatment groups, control group also significantly rises It is high；As shown in Figure 4 C, there is notable positive correlation between GPR120 gene expressions and cell eGFP mean fluorescence intensities, with The increase of eGFP mean fluorescence intensities, the rise of GPR120 gene expression doses.

Claims (5)

  1. Applications of the 1.eGFP in the detection of GPR120 gene expression doses.
  2. 2. application according to claim 1, it is characterised in that the eGFP and GPR120 gene expression doses it is linear Relation, i.e., with the increase of eGFP fluorescence average intensity values, the increase of GPR120 gene expression doses.
  3. 3. the detection method of the GPR120 gene expression doses based on eGFP, is comprised the following steps that：
    Step 1, eGFP fragments are pinpointed to the terminator codon for being inserted into GPR120 genes by CRISPR/Cas9 technologies first At TAA, eGFP is expressed and is expressed with GPR120, obtain the transgenic models mouse that eGFP marks GPR120 positive cells；
    Step 2, then positive cell fluorescence intensity of the application fluorescence analyser to mouse under 488nm laser excitations is surveyed It is fixed.
  4. 4. detection method according to claim 3, it is characterised in that the fluorescence intensity of the step 2 passes through unicellular flat Equal fluorescence intensity is expressed.
  5. 5. detection method according to claim 3, it is characterised in that the specific steps of described step 2：
    Step 2.1, the laser intensity index for carrying out fluorescence analyser using the mouse cell do not demarcated by eGFP is corrected；
    Step 2.2, in take-up step 1 different tissues of transgenic models mouse eGFP positive cells, by through step 2.1 school Fluorescence analyser after just carries out fluorescent strength determining, obtains the relation between eGFP and GPR120 gene expression doses；Simultaneously The gene expression dose of GPR120 in eGFP positive cells, checking eGFP and GPR120 gene expressions are detected by RT-PCR method Relation between level.
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