CN115216492A - Preparation method and application of mouse primary glioma model - Google Patents
Preparation method and application of mouse primary glioma model Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a mouse primary glioma model, which comprises the following steps: 1) Construction of a PB-CAG-MCS-IRES-EGFP plasmid, 2) construction of a PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting a Pdgfa gene into a polyclonal site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing PDGFA protein carrying an HA label and green fluorescent protein EGFP for tracing; 3) And preparing a primary glioma mouse model to obtain the primary glioma mouse model. The invention provides an important research model for the related researches such as the generation and treatment of glioma.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of a mouse primary glioma model.
Background
Gliomas (gliomas) are common tumors of the central nervous system. Glioblastoma (GBM) is even more highly malignant tumor of the nervous system. Glioblastoma GBM can be classified into Proneural (PN), mesenchymal (MES), classical (CL) and Neural (NL) types according to its characteristics such as gene expression. Different tumor subtypes are often associated with different gene mutations. For example, over 90% of CL-GBMs are denied enhanced EGFR activity and CDKN2A loss, MES-GBMs are often associated with NF1/PTEN gene mutations, while PDGFRA amplification and IDH1 gene mutations are observed in the vast majority of PN-GBMs. The current research model for glioma mainly comprises glioma cells cultured in vitro, such as A172, U-251 and the like; or a transplanted tumor formed by inoculating in vitro cultured tumor cells into the mouse brain. However, these models can only study the progress after tumor formation, therapeutic effects, and other problems, and cannot study the problem of tumor occurrence, and the tumor cells in these cell lines often have a great difference from the in vivo primary glioma. Therefore, the primary glioma model is an indispensable tool for glioma research and drug screening.
At present, the preparation of the primary glioma model mainly depends on the use of a plurality of genetically modified mice, and the preparation difficulty is large. The following methods are common: (1) Tool mice expressing Cre recombinase through NG2-creER, hGFAP-creER, nestin-Cre and the like bind to Pten fx/fx Or p53 fx/fx LSL-EGFRviii and other mouse strains, conditional knock-out of genes such as p53, nf1 and Pten or activation of EGFR and PDGFRa signaling pathway in Oligodendrocyte Precursor Cells (OPCs) and neural precursor cells; (2) By means of Olig2-TVA, hGFAP-TVA and Nestin-TVA tool mice for expressing avian virus receptor A (TVA), through injecting avian virus RCAS plasmid carrying exogenous genes into mouse brain, replication and infection of avian viruses in mouse nerve cells are realized, continuous expression of the exogenous genes is mediated, and glioma is induced finally; (3) These were achieved by electrotransfer of plasmids expressing small guide RNAs against the p53, nf1 and Pten genes, with the aid of transgenic mice expressing Cas9Inactivation of cancer suppressor gene to induce mouse primary glioma; (4) The transposon system is also a recently adopted method, and can induce glioma by transposing and integrating an exogenous gene into a specific genetically modified mouse brain nerve cell to activate or knock out a specific gene. However, these primary glioma models are based on genetically modified mice and are therefore subject to practical limitations on raw materials. Therefore, there is a need to develop a primary glioma model that can be achieved in normal wild-type mice.
The piggyBac transposon system is also proved to be capable of effectively realizing the stable expression of the exogenous gene in mouse cells, has higher efficiency than other transposon systems, and is an ideal method for expressing the exogenous gene in mouse tissues or knocking out genes. It has been shown that the aforementioned PN-GBM-like glioma can be induced in the mouse brain by activating the continuous expression of the Pdgfa gene alone.
Disclosure of Invention
The invention provides a preparation method and application of a mouse primary glioma model, which aim to solve the problems in the prior art.
The scheme of the invention is as follows:
a preparation method of a mouse primary glioma model comprises the following steps:
1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a polyclonal site is added between the promoter and the IRES-EGFP sequence;
2) The construction of a PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, wherein a Pdgfa gene is inserted into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid to express a PDGFA protein carrying an HA label and a green fluorescent protein EGFP for tracing;
3) The preparation of the primary glioma mouse model comprises the steps of inducing a mouse by using a plasmid PB-CAG-Pdgfa-HA-IRES-EGFP and a plasmid pCAGGS-piggyBac Transposase for expressing Transposase together, and obtaining the primary glioma mouse model.
Preferably, the promoter is a CAG promoter or a promoter available for other mammalian cells.
As a preferred technical scheme, the PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is integrated into a cell genome in the presence of piggyBac transposase to realize the stable expression of a target gene in a transferred cell and a progeny cell thereof.
The invention also discloses a preparation method of the mouse primary glioma model, and piggyBac transposon plasmid PB-CAG-MCS-IRES-EGFP is used in the preparation method, and the nucleotide sequence of the PB-CAG-MCS-IRES-EGFP is shown in SEQ ID No. 1.
The invention also discloses an application of the preparation method of the mouse primary glioma model, and provides an important research model for the development and treatment research of glioma.
Due to the adoption of the technical scheme, the preparation method of the mouse primary glioma model comprises the following steps: 1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a polyclonal site is added between the promoter and the IRES-EGFP sequence; 2) The construction of PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into the multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing PDGFA protein carrying HA tags and green fluorescent protein EGFP for tracing; 3) Preparing a primary glioma mouse model, and inducing a mouse by using a plasmid PB-CAG-Pdgfa-HA-IRES-EGFP and a plasmid pCAGGS-piggyBac Transposase for expressing Transposase together to obtain the primary glioma mouse model.
The invention has the advantages that:
the invention constructs a piggyBac transposon plasmid carrying EGFP, and a PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is obtained by inserting a Pdgfa gene into the plasmid, and is transferred into the cerebral cortex of a mouse at an embryonic stage of 14.5 (E14.5) by a mouse embryo plasmid electrotransfer technology, and primary glioma is induced after the mouse is born. By using the plasmid system constructed by the invention and the method described, primary glioma can be induced in the brain of a common wild-type mouse without the need for various genetically modified mice. The invention provides an important research model for the related researches such as the generation and treatment of glioma.
Drawings
FIG. 1 is a table diagram of a model of Pdgfa-induced primary glioma in a mouse according to an embodiment of the present invention;
FIG. 2 is a graph showing the expression of each marker in the primary glioma detected by immunohistochemistry and in situ hybridization according to an embodiment of the present invention;
FIG. 3 is a PB-CAG-MCS-IRES-EGFP plasmid map of the present invention.
Detailed Description
In order to make up for the above deficiencies, the present invention provides a method for preparing a mouse primary glioma model and applications thereof to solve the above problems in the background art.
A method for preparing a mouse primary glioma model comprises the following steps:
1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a polyclonal site is added between the promoter and the IRES-EGFP sequence;
2) The construction of PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into the multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing PDGFA protein carrying HA tags and green fluorescent protein EGFP for tracing;
3) The preparation of the primary glioma mouse model comprises the steps of inducing a mouse by using a plasmid PB-CAG-Pdgfa-HA-IRES-EGFP and a plasmid pCAGGS-piggyBac Transposase for expressing Transposase together, and obtaining the primary glioma mouse model.
The promoter is CAG promoter or other promoter available for mammalian cells.
The PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is integrated into a cell genome in the presence of piggyBac transposase, so that stable expression of a target gene in a transferred cell and a progeny cell thereof is realized.
The invention also discloses a preparation method of the mouse primary glioma model, which uses piggyBac transposon plasmid PB-CAG-MCS-IRES-EGFP, and the nucleotide sequence of PB-CAG-MCS-IRES-EGFP is shown in SEQ ID No. 1. The plasmid contains a CAG promoter and an IRES-EGFP sequence at the downstream of the CAG promoter, and a multiple cloning site is added between the CAG promoter and the IRES sequence for inserting a foreign gene. The plasmid contains a nucleic acid sequence required by piggyBac transposase mediated transposition, and can realize stable expression of a target gene in transferred cells and progeny cells thereof.
The plasmid pCAGGS-piggyBac Transposase for expressing piggyBac Transposase is named pCAGGS-PBase.
Through In Ovo Electroporation (IOE) of mice, the plasmid PB-CAG-Pdgfa-HA-IRES-EGFP and the plasmid pCAGGS-piggyBac Transposase expressing Transposase are jointly electroporated to the cerebral cortex of the mice in the embryonic period, thereby inducing the mice to generate primary glioma after birth.
The invention is based on a transposon gene delivery system, and realizes continuous overexpression of Pdgfa in mouse brain by an embryo electrotransformation method, thereby obtaining a mouse primary glioma model. Under the technical conditions of using the transposon system, the replacement of CAG promoter in the invention for other available promoters of mammalian cells, or the adjustment of the mouse development period of embryo electrotransfer, such as embryonic period 12 days (E12) to postnatal period 1 week (P7) are all in the scope of the claims of the present invention.
The invention also discloses an application of the preparation method of the mouse primary glioma model, and provides an important research model for the development and treatment research of glioma.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example (b):
construction of PB-CAG-MCS-IRES-EGFP plasmid:
the CAG-MCS-IRES-EGFP fragment needs to be obtained firstly. This fragment contains both the CAG promoter and the IRES-EGFP fragment. By the technique of overlap extension PCR, two DNA fragments can be spliced together. Wherein, the CAG promoter fragment is from pCAGGS-mCherry plasmid, and the IRES-EGFP fragment is from pCIG2-Flag-mNuak1 plasmid. The primers were designed as follows:
CAG-IRES-F:
5-gccaccatgtgtacagaactcgagtttaaatcggatccgatccgcccctctccctcccccccccctaacgttact-3
CAG-IRES-R:
5-ctcgagttctgtacacatggtggctgacatctgatgatggctagccccgggcccgcggtaccgtcgact-3
CAG-F:5-GGACTAGTtattaatagtaatcaattacggggtcattagttcatagcccat-3EGFP-R:
5-GCACGCGTCGACtgtaccgctcatctgttagtggtgcctagattacttgtagagctcgtccatgcc-3
PCR-1# amplification was performed using primers CAG-F and CAG-IRES-R as a template for plasmid pCAGGS-mCherry (Addgene, # 41583) and the target product was about 1.7kb. PCR-2# amplification was performed using primers CAG-IRES-F and EGFP-R as primers and plasmid pCIG 2-Flag-mNeak 1 (Addgene, # 168507) as a template, and the target product was about 1.4kb. The 2 PCRs were all 30uL systems, 5 cycles. As the enzyme, primeSTAR GXL DNA Polymerase (# R050Q) from TAKARA was used, and DNA Polymerase instructions were referred to for the amplification system. And after the PCR-1# and the PCR-2# are finished, performing a third PCR reaction by taking the two PCR products as templates (the dosage is 1uL/30uL system) and the CAG-F and the EGFP-R as primers, namely, performing PCR-3#. The PCR reaction was as above, but the amplification was 60uL, the cycle number was set to 30, and the target fragment size was about 3.2kb. After the amplification, the PCR-3# product was subjected to 1% agarose gel electrophoresis, and then the target fragment was purified by a gel recovery kit (50 uL water elution product). The DNA fragment is CAG-MCS-IRES-EGFP. The target fragment was digested with SpeI and SalI (enzymes from Thermo, # FD1254, # FD 0644), the system was 50uL, the product was recovered from gel 43uL, buffer 5uL, and both enzymes were incubated at 37 ℃ overnight at 1uL each. And recovering the PCR product subjected to enzyme digestion to 30uL deionized water by using the DNA Clean-Up kit for later use.
Thereafter, it is necessary to obtain a plasmid fragment that can be recognized by transposase. Primers were designed as follows:
PB-F:5-GCACGCGTCGACaatcaacctctggattacaaaatttgtg-3
PB-R:5-GGACTAGTggccttggaggccttttccccgtatccccccag-3
primers PB-F and PB-R are used for PCR, and a template is a plasmid PB-CMV-MCS-EF1 alpha-GFP-Puro (SBI, # PB 513B-1). The size of the PCR product fragment was about 4.9kb, and the reaction system was the same as above. After agarose gel electrophoresis of the PCR product, the product is purified and recovered, and then is subjected to SpeI and SalI double digestion. The subsequent fragment recovery method is the same as above. Connecting the fragment subjected to double enzyme digestion with the CAG-MCS-IRES-EGFP fragment: 3.2kb double digestion fragment 3uL, CAG-MCS-IRE-EGFP double digestion fragment 5.5uL, ligase Buffer 1uL, T4 Ligase 0.5uL. Ligation was performed at room temperature for 30min to transform E.coli DH5a competent cells. The next day, monoclonal colonies were picked for sequencing. The clone with correct sequencing can be inoculated and cultured, and then the plasmid is extracted. Thus obtaining the PB-CAG-MCS-IRES-EGFP plasmid. This plasmid introduces a multiple cloning site between the CAG promoter and IRES, contains NheI and BsrGI restriction enzyme recognition sites, allowing for the insertion of subsequent foreign gene fragments (fig. 3).
Construction of PB-CAG-Pdgfa-HA-IRES-EGFP plasmid:
to obtain Pdgfa fragments, primers were designed as follows:
Pdgfa-F:5-GCTCTAGAGCCACCatgaggacctgggcttgcctgctg-3
Pdgfra-R:
5-GCGCTGGTACCttatgcataatcaggcacatcgtaaggatacctcacatctgtctcctcctcccg-3
the PCR method, the DNA fragment recovery method, the double digestion and purification method of the DNA fragment are the same as those described above. The template used for PCR is cDNA obtained by reverse transcription of total RNA extracted from mouse brain tissue 2 weeks after birth, and the size of the target fragment is about 630bp.
Then, vector PB-CAG-MCS-IRES-EGFP was subjected to NheI and BsrGI double digestion, that is, plasmid 5ug,50uL system, endonuclease was 2.5uL each, and incubated at 37 ℃ overnight. The 1% agarose gel was electrophoresed, and the target fragment of about 8kb (50 uL water eluted product) was purified by a gel recovery kit. This fragment was ligated with the aforementioned Pdgfa-HA fragment: vector fragment 1uL, pdgfa-HA double digestion fragment 5uL, deionized water 2.5uL, ligase Buffer 1uL, T4 Ligase 0.5uL. Ligation was performed at room temperature for 30min to transform E.coli DH5a competent cells. The next day, monoclonal colonies were picked for sequencing. The clone with correct sequencing can be inoculated and cultured, and then the plasmid is extracted. The plasmid PB-CAG-Pdgfa-HA-IRES-EGFP is obtained, and the plasmid can simultaneously express green fluorescent protein EGFP and PDGFA protein carrying HA labels under the drive of a CAG promoter.
3. Preparation of primary glioma mouse model:
plasmid PB-CAG-Pdgfa-HA-IRES-EGFP is extracted from escherichia coli by a plasmid extraction kit for removing endotoxin, and the plasmid PB-CAG-Pdgfa-HA-IRES-EGFP is dissolved in deionized water without endotoxin to ensure that the concentration of the plasmid is 5000 ng/mu l. Female and male adult mice were placed in the same cage in the evening, separated the following morning, and scored as embryonic age 0.5 days (E0.5) by midday. E14.5 pregnant mice were taken for mouse embryo plasmid electrotransformation. Detailed experimental procedures were performed as reported in the literature. The general flow is as follows: after anaesthetizing pregnant mice, sterilizing the abdominal surface, cutting off abdominal hair, opening the abdominal cavity, pulling out the uterus of mice containing embryos, injecting 2-3uL of plasmid mixed with FastGreen dye with the final concentration of 0.01% into the lateral ventricle of the embryo through a microinjection instrument, placing circular electrodes with the diameter of 6cm on both sides of the brain of the embryo, and applying 45V pulse voltage (50 ms each time, 950ms each time interval, 5 times of continuous electric shock); the uterus was carefully moved back to the mouse uterus, the wound was sutured, and after sterilization, the body temperature was maintained at 37 ℃ until the mice recovered. Mouse pups were born approximately 4-5 days later, and after birth, mice were examined for glioma formation when they survived to 1 month of age. Control mice electrotransfer plasmid PB-CAG-MCS-IRES-EGFP.
The results showed that one month after birth, mice were misshapen in head, severely impaired in mobility, and also smaller in size than control mice (fig. 1A-C). After dissection, the brain tissue was subjected to slice analysis, and the results showed that: the volume of the tumor site in the cerebral cortex of mice is equivalent to the size of normal brain tissue, and in situ hybridization experiments on Pdgfa mRNA confirm that the tumor formation region has extensive high expression of Pdgfa gene (figure 1D, E). Immunofluorescence and immunohistochemical staining showed that cells at the tumor site highly expressed markers for PN-GBM such as OLIG2, PDGFRA, SOX10, etc. (fig. 2a, B, a ', B'). Meanwhile, in the tumor region, GFAP +/Glast + astrocytes were absent (fig. 2D), few NeuN + neurons were present (fig. 2C), but some number of IBA1+/CD68+ activated microglia were present (fig. 2e, D). These results indicate that the mouse primary glioma model was successfully constructed using this method. The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Zhejiang European Tess Biotech Co., ltd
<120> preparation method and application of mouse primary glioma model
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8070
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 60
tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 120
tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 180
acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 240
aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 300
cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 360
cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 420
cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca 480
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 540
gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 600
aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc 660
agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg ctcgacacgc 720
tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg tacgttaaag 780
ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat 840
taatttgaat agatattaag ttttattata tttacactta catactaata ataaattcaa 900
caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc 960
tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat 1020
tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc 1080
gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg 1140
cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg 1200
ggatacgggg aaaaggcctc caaggccact agttattaat agtaatcaat tacggggtca 1260
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 1320
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 1380
acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac 1440
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 1500
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 1560
tacatctacg tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt 1620
cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 1680
attttgtgca gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg 1740
gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc 1800
tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 1860
cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc 1920
gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 1980
cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc 2040
tgcgtgaaag ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg 2100
ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct 2160
gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc 2220
ggccgggggc ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg 2280
ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc 2340
cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac 2400
ggggcgtggc gcggggctcg ccgtgccggg cggggggtgc cggcaggtgg gggtgccggg 2460
cggggcgggg ccgcctcggg cctgggaggg ctcgggggag gggcgcggcg gcccccggag 2520
cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga 2580
gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg 2640
ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg 2700
cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg 2760
ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg 2820
gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct 2880
acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcct 2940
cgagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccggggctag 3000
ccatcatcag atgtcagcca ccatgtgtac agaactcgag tttaaatcgg atccgatccg 3060
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 3120
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 3180
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 3240
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 3300
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 3360
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 3420
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 3480
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta 3540
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 3600
ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gtgagcaagg 3660
gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 3720
gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 3780
tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 3840
tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 3900
tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 3960
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 4020
agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 4080
actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 4140
acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 4200
agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 4260
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 4320
tgaccgccgc cgggatcact ctcggcatgg acgagctcta caagtaatct aggcaccact 4380
aacagatgag cggtacagtc gacaatcaac ctctggatta caaaatttgt gaaagattga 4440
ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 4500
tgtatcagtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4560
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4620
caatgtatct tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct 4680
atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc 4740
caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca 4800
caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat 4860
tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc 4920
tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg 4980
ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc 5040
cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct 5100
ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac 5160
gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt 5220
aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac 5280
ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat 5340
gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt 5400
caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg 5460
gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga 5520
agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg 5580
ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc 5640
catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg 5700
gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac 5760
cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg 5820
atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc 5880
ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga 5940
aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 6000
tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 6060
cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 6120
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6180
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6240
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6300
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6360
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6420
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6480
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6540
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6600
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6660
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 6720
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 6780
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6840
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6900
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6960
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 7020
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 7080
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 7140
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 7200
agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 7260
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 7320
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 7380
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 7440
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 7500
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7560
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7620
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7680
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 7740
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 7800
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7860
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7920
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7980
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 8040
ccgcgcacat ttccccgaaa agtgccacct 8070
<210> 2
<211> 615
<212> DNA
<213> Mus musculus
<400> 2
atgaggacct gggcttgcct gctgctcctc ggctgcggat acctcgccca tgccctggcc 60
gaggaagccg agataccccg ggagttgatc gagcggctgg ctcgaagtca gatccacagc 120
atccgggacc tccagcgact cttggagata gactccgtag gggctgagga tgccttggag 180
acaagtctga gagcccatgg gtcccatgcc attaaccatg tgcccgagaa gcggcctgtg 240
cccattcgca ggaagagaag tattgaggaa gccattcctg cagtttgcaa gaccaggacg 300
gtcatttacg agatacctcg gagccaggtg gaccccacat cggccaactt cctgatctgg 360
cccccatgtg tggaggtgaa gcgctgcact ggctgttgta acaccagcag cgtcaagtgc 420
cagccttcac gggtccacca ccgcagtgtc aaggtggcca aagtggagta tgtcaggaag 480
aagccaaaat tgaaagaggt ccaggtgagg ttagaggaac acctggagtg tgcatgtgcg 540
acctccaacc tgaacccaga ccatcgggag gaggagacag atgtgaggta tccttacgat 600
gtgcctgatt atgca 615
Claims (5)
1. A preparation method of a mouse primary glioma model is characterized by comprising the following steps:
1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a polyclonal site is added between the promoter and the IRES-EGFP sequence;
2) The construction of a PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, wherein a Pdgfa gene is inserted into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid to express a PDGFA protein carrying an HA label and a green fluorescent protein EGFP for tracing;
3) Preparing a primary glioma mouse model, and inducing a mouse by using a plasmid PB-CAG-Pdgfa-HA-IRES-EGFP and a plasmid pCAGGS-piggyBac Transposase for expressing Transposase together to obtain the primary glioma mouse model.
2. The method of claim 1, wherein the method comprises the steps of: the promoter is CAG promoter or other promoter available for mammalian cells.
3. The method for preparing a mouse primary glioma model of claim 1, wherein: the PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is integrated into a cell genome in the presence of piggyBac transposase, so that stable expression of a target gene in a transferred cell and a progeny cell thereof is realized.
4. The method of claim 1, wherein piggyBac transposon plasmid PB-CAG-MCS-IRES-EGFP is used for the preparation of a primary mouse glioma model, wherein: the nucleotide sequence of the PB-CAG-MCS-IRES-EGFP is shown as SEQ ID No. 1.
5. Use of a method of preparing a model of primary mouse glioma according to claims 1 to 3, characterized in that: provides an important research model for the development and treatment research of glioma.
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