CN103074346A - Method for establishing finite cell strain for screening read-though promoting drug - Google Patents
Method for establishing finite cell strain for screening read-though promoting drug Download PDFInfo
- Publication number
- CN103074346A CN103074346A CN2013100233844A CN201310023384A CN103074346A CN 103074346 A CN103074346 A CN 103074346A CN 2013100233844 A CN2013100233844 A CN 2013100233844A CN 201310023384 A CN201310023384 A CN 201310023384A CN 103074346 A CN103074346 A CN 103074346A
- Authority
- CN
- China
- Prior art keywords
- pdsred
- egfpmtag
- expression vector
- cell
- egfp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a eukaryote expression vector for expressing a green fluorescent protein (EGFP) and a red fluorescent protein (DsRed); a mutant contains a premature termination condon in a 445 amino acid position of the green fluorescent protein EGFP; and the expression of the green fluorescent protein EGFP can be effectively stopped by a mutational site. A constructed double fluorescent expression vector and a mutant thereof are introduced into an African green monkey kidney cell Cos-7 and treated by a read-though promoting drug PTC124; expression intensities of the green fluorescent protein and the red fluorescent protein are detected by using a flow cytometry; and the read-though promoting efficiency of the read-though promoting drug to the vector can be efficiently detected. In addition, different nonsense mutations and upstream and downstream correlation sequences can be introduced into a multiple clone site of the expression vector, so that targeted drugs for diseases initiated by the different nonsense mutations can be specifically screened.
Description
Technical field:
The present invention relates to biotechnology cell lines field, be specifically related to a kind ofly for the short establishment method of reading over the limited cell strain of drug screening, say more specifically the establishment method of a kind of pair of fluorescence mammalian expression vector and the limited cell strain of PTC mutant thereof and two fluorescence mammalian expression vectors and the limited cell strain of PTC mutant thereof of setting up according to the method.
Background technology:
According to human genome mutation database (Human Genome Mutation Database, HGMD, http://www.hgmd.org) 2012 authority's statistics, because phase shift mutation, alternative splicing or directly nonsense mutation, formed premature termination codon (Premature termination codons at human genome, PTC), the existence of PTC makes body produce the protein of Truncated or the protein with negative dominant effect, has human inheritance's disease of 1/3rd to cause thus; Human inheritance's disease of 11.2% directly causes owing to nonsense mutation or follows appearance.
One of main direction of clinical treatment genetic diseases is exactly by the short of PTC readed over, thereby suppresses to produce Truncated protein, and the activated protein that promotes to produce total length is alleviated and treated genetic diseases.
At present in the gene PTC to read over research be one of focus of molecular cytobiology, the treatment of the genetic diseases that causes is thus had potential using value.Mainly for genetic diseases cystic fibrosis (cystic fibrosis is arranged, CF), Du Shi muscular dystrophy (Duchenne muscular dystrophy, DMD), hemophilia A and B(hemophilia A, HA andB, HB), familial benign pemphigus (Hailey-Hailey disease, HHD) and mucopolysaccharidosis (mucopolysaccharidosis, MPS).
The short PTC of medicine reads over the mechanism research that expression also is used for cancer, tumour and part genetic diseases that the gene nonsense mutation causes, and has obtained considerable progress, has obtained a series of significant molecules data and test-results.
Studies show that, aminoglycoside (for example gentamicine) compound can be attached to ribosomal identification center (decodingcenter), reduce the accuracy of codon and anticodon pairing, cause mispronouncing of codon among the mRNA, in the zooblast of cultivating and human body cell, all obtained the confirmation of experiment; Compound P TC124, from different on the aminoglycoside medicaments structure, and the short efficient of reading over is very high, is used for for three phases clinical, trade(brand)name Ataluren.Lack at present high-throughout cell levels drug screening system both at home and abroad, therefore be badly in need of setting up corresponding cell strain for external screening because the potential drug of the disease that nonsense mutation causes more can be used for screening because the clinical treatment medicine of the heredopathia that nonsense mutation causes.
Summary of the invention:
The present invention is intended to address the above problem, and provides a kind of for the short establishment method of reading over the limited cell strain of drug screening, and two fluorescence mammalian expression vectors and the limited cell strain of PTC mutant thereof set up according to the method.
For achieving the above object, the invention provides following technical scheme:
A kind of pair of fluorogene, its nucleotides sequence are classified SEQ ID NO:1 as.
A kind of mammalian expression vector contains above-mentioned pair of fluorogene, carrier called after pDsRed-EGFPmtag-.
A kind of for the short establishment method of reading over the limited cell strain of drug screening, comprise the steps:
1) utilize gene recombination technology with DsRed and EGFP gene clone to the pEGFP-N1 mammalian expression vector, be built into pDsRed-EGFP, recombinant plasmid is cut evaluation through enzyme;
2) utilize PCR Fast Fixed-point mutation method, the terminator codon tag of DsRed gene is suddenlyd change to tac, and adjust DsRed and EGFP gene to a reading frame, be built into two fluorescent expression vector pDsRed-EGFPmtag-;
3) utilize the method for PCR rite-directed mutagenesis, 445 amino acid positions of green fluorescent protein EGFP are sported contain a premature termination codon, obtain the mutant pDsRed-EGFPmtag-Y445X of two fluorescence mammalian expression vectors;
4) cultivate African green monkey kidney cell Cos-7, substratum is DMEM, adds 10% foetal calf serum, does not add any microbiotic and cultivates, and trypsin digestion cell is by 2 * 10
5Cell/mL density kind is in 6 orifice plates;
5) utilize liposome Lipofectamine
TM2000 with plasmid pDsRed-EGFPmtag-and pDsRed-EGFPmtag-Y445X difference transfection Cos-7 cell, and transfection was changed fresh medium and obtained limited cell strain after 8 hours.
Process limited cell strain with urging to read over medicine PTC124, after 48 hours, use the confocal laser scanning microscope cell, loading flow cytometer, the short efficient of reading over of real-time quantitative PCR detection of drugs.
The limited cell strain that the present invention sets up contains two fluorescence mammalian expression vector pDsRed-EGFPmtag-and PTC mutant pDsRed-EGFPmtag-Y445X thereof.Two fluorescence mammalian expression vectors and the limited cell strain of PTC mutant thereof of building system, method and foundation of the present invention can be used for the screening that nonsense mutation causes the medicine of genetic diseases, screening that PTC reads over medicine etc.The limited cell strain set up of the present invention also can be used for the inhibition of NMD approach and the short PTC of medicine are readed over the mechanism research of cancer, tumour and the part genetic diseases expressing and then cause for the gene nonsense mutation in addition.
Description of drawings:
Fig. 1 is that two fluorescence mammalian expression vector pDsRed-EGFPmtag-form schematic diagram
Fig. 2 is two fluorescence mammalian expression vector pDsRed-EGFPmtag-evaluation figure, and wherein: swimming lane 1 is blank; Swimming lane 2 is identified for PCR; Swimming lane 3 is identified for double digestion
Fig. 3 is two fluorescence mammalian expression vector pDsRed-EGFPmtag-order-checking evaluation figure
Fig. 4 is two fluorescence mammalian expression vector PTC mutant pDsRed-EGFPmtag-Y445X order-checking evaluation figure
Fig. 5 is lower pair of fluorescence mammalian expression vector of fluorescent microscope and PTC mutant photo thereof, wherein: E is the A(Cos-7-pDsRed-EGFPmtag-green fluorescence) and the C(Cos-7-pDsRed-EGFPmtag-red fluorescence) the stack photo, F is the B(Cos-7-pDsRed-EGFPmtag-Y445X green fluorescence) and the D(Cos-7-pDsRed-EGFPmtag-Y445X red fluorescence) the stack photo
Fig. 6 is the streaming figure of limited cell strain Cos-7-pDsRed-EGFPmtag-of the present invention.
Fig. 7 is limited cell strain Cos-7-pDsRed-EGFPmtag-of the present invention and the stream data of PTC mutant Cos-7-pDsRed-EGFPmtag-Y445X after different concns PTC124 processes thereof.
Fig. 8 is limited cell strain Cos-7-pDsRed-EGFPmtag-of the present invention and the fluorescence real-time quantitative PCR data of PTC mutant Cos-7-pDsRed-EGFPmtag-Y445X after different concns PTC124 processes thereof.
Embodiment:
1) structure of the two fluorescence mammalian expression vectors of restructuring
Utilize Xho I and EcoR I that the DsRed fluorescence protein gene is carried out the gene fragment that double digestion obtains 0.8kb, electrophoresis reclaims.Mammals efficient expression vector pEGFP-N1 also uses Xho I and the linear carrier of EcoR I double digestion, and electrophoresis reclaims.Use the T4 dna ligase to be connected itself and the DsRed fluorescence protein gene fragment that reclaims, obtain recombinant plasmid pDsRed-EGFP, recombinant plasmid is cut evaluation through enzyme.(see figure 2)
Design the mutant primer of a pair of complete complementary, use the high-fidelity enzyme to carry out pcr amplification with recombinant plasmid pDsRed-EGFP as template, product is through Dpn I digestion template, get 10 μ L product transform bacterias, the picking clone extracts plasmid, (see figure 3) is identified in order-checking, and two fluorescence mammalian expression vector pDsRed-EGFPmtag-(that obtain recombinating see Fig. 1).
2) structure of the two fluorescence mammalian expression vector PTC mutant of restructuring
Design the mutant primer of a pair of complete complementary, use the high-fidelity enzyme to carry out pcr amplification with recombinant plasmid pDsRed-EGFPmtag-as template, product is through Dpn I digestion template, get 10 μ L product transform bacterias, the picking clone extracts plasmid, (see figure 4) is identified in order-checking, obtains PTC mutant plasmid pDsRed-EGFPmtag-Y445X.
3) cultivation of mammalian cell Cos-7 with go down to posterity
Cell derived is in Shanxi university student's thing technical institute, and recovery cell cultures well is in 25cm
2In the Tissue Culture Flask, 37 ℃, 5%CO
2, substratum is DMEM, adding foetal calf serum to final concentration is 10%.Treating that cell degree of converging reaches more than 70% can go down to posterity, discard substratum, wash 2 times with PBS, discard PBS, add 0.25% trysinization liquid 1mL, with culturing bottle as for 37 ℃ of incubator preheatings, treat cell rounding but not yet discard pancreatin during fall in flakes, add the DMEM contain 10% foetal calf serum and stop digestion, and blow and beat gently cell and make it to come off, then be sub-packed in 3 culturing bottles according to 1:3 and add an amount of culture medium culturing.
4) transfection of Cos-7 cell and the short drug treating of reading over
A, transfection first day press 2 * 10 with the Cos-7 cell
5Cell/mL density kind in 6 orifice plates, incubated overnight so that next day cell degree of converging reach 90-95%.
B, transfection second day, the two fluorescence mammalian expression vectors of preparation and PTC mutant plasmid and liposome mixed solution are used for the Cos-7 cell of cultivating in transfection 6 well culture plates in aseptic centrifuge tube.Solution A: 4 μ g plasmid DNA are dissolved in the 250 μ L serum free mediums.Solution B: 10 μ L Lipofectamine
TM2000 are dissolved in the 250 μ L serum free mediums room temperature 5-25 minute.Mixed solution A and solution B, mixing gently, room temperature is more than 20 minutes.The solution 500 μ L of mixing are added in the cell culture medium of 6 well culture plates mixing gently, 37 ℃, 5%CO
2Cultivated 24 hours.
The short drug treating of reading over of c, transfectional cell
After the transfection 8 hours, change fresh substratum, add medicine PTC124 and intervene, activity is respectively 1,2,4,8,16,32,64nmol/ml.
The results of d, cell
After 48 hours, the laser confocal microscope microscopy, Cos-7-pDsRed-EGFPmtag-has obvious redness (seeing Fig. 5 A) and green fluorescence (seeing Fig. 5 C) to express, and is yellow (seeing Fig. 5 E) after the stack, and the Cos-7-pDsRed-EGFPmtag-Y445X green fluorescence obviously weakens (seeing Fig. 5 B).Discard substratum, wash 2 times with PBS, discard PBS, add 0.25% trysinization liquid 1mL, culturing bottle as for the preheating of 37 ° of C incubators, is treated cell rounding but not yet discarded pancreatin during fall in flakes, add the DMEM contain 10% foetal calf serum and stop digestion, and blow and beat gently cell and make it to come off.
5) check of the flow cytometry of two fluorescence mammalian expression vectors and the limited cell strain of PTC mutant thereof and real-time quantitative PCR detect.
The Flow cytometry of a, two fluorescence mammalian expression vector and the limited cell strain of PTC mutant thereof
After the cell of results is used the physiological saline Eddy diffusion, detect (see figure 6) at flow cytometer, with red fluorescence as internal reference, data analysis is found, PTC124 has effectively promoted reading over of two fluorescence mammalian expression vector PTC mutant, and the short efficient of reading over of PTC124 has dose-effect relationship within the specific limits, under the concentration of 4nmol/ml, urge to read over most effective, the limited cell strain that two fluorescence mammalian expression vectors and PTC mutant thereof are described is readed over the screening system as short nonsense mutation, can high-throughout screening drug candidate (see figure 7).
The real-time quantitative PCR of b, two fluorescence mammalian expression vector and the limited cell strain of PTC mutant thereof detects
According to green fluorescent protein EGFP gene order, design a pair of specificity real-time quantitative PCR primer, with the Cos-7 cell of untransfected as negative control, the cDNA that becomes take the mRNA reverse transcription of limited cell strain Cos-7-pDsRed-EGFPmtag-and Cos-7-pDsRed-EGFPmtag-Y445X is as template, carry out the real-time quantitative PCR reaction, result and the Flow cytometry (see figure 8) that comes to the same thing illustrates that PTC124 has effectively promoted reading over of two fluorescence mammalian expression vector PTC mutant.
Claims (3)
1. two fluorogene, its nucleotides sequence is classified SEQ ID NO:1 as.
2. a mammalian expression vector contains claimed in claim 1 pair of fluorogene.
3. one kind is used for the short establishment method of reading over the limited cell strain of drug screening, comprises the steps:
1) utilize gene recombination technology with DsRed and EGFP gene clone to the pEGFP-N1 mammalian expression vector, be built into pDsRed-EGFP, recombinant plasmid is cut evaluation through enzyme;
2) utilize PCR Fast Fixed-point mutation method, the terminator codon tag of DsRed gene is suddenlyd change to tac, and adjust DsRed and EGFP gene to a reading frame, be built into two fluorescence mammalian expression vector pDsRed-EGFPmtag-;
3) utilize the method for PCR rite-directed mutagenesis, 445 amino acid positions of green fluorescent protein EGFP are sported contain a premature termination codon, obtain the mutant pDsRed-EGFPmtag-Y445X of two fluorescence mammalian expression vectors;
4) in substratum DMEM, add 10% foetal calf serum, cultivate African green monkey kidney cell Cos-7, then add trypsin digestion cell, by 2 * 10
5Cell/mL density kind is in 6 orifice plates;
5) utilize liposome Lipofectamine
TM2000 with plasmid pDsRed-EGFPmtag-and pDsRed-EGFPmtag-Y445X difference transfection Cos-7 cell, and transfection was changed fresh culture and obtained limited cell strain after 8 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100233844A CN103074346A (en) | 2013-01-22 | 2013-01-22 | Method for establishing finite cell strain for screening read-though promoting drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100233844A CN103074346A (en) | 2013-01-22 | 2013-01-22 | Method for establishing finite cell strain for screening read-though promoting drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103074346A true CN103074346A (en) | 2013-05-01 |
Family
ID=48151041
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100233844A Pending CN103074346A (en) | 2013-01-22 | 2013-01-22 | Method for establishing finite cell strain for screening read-though promoting drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103074346A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106979938A (en) * | 2017-03-09 | 2017-07-25 | 东华大学 | It is a kind of to carry out the method that destination gene expression is quantitatively detected using flow cytometer |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001038557A1 (en) * | 1999-11-24 | 2001-05-31 | The University Of New South Wales | Method of screening multiply transformed cells using bicistronic expression of fluorescent proteins |
| CN101260408A (en) * | 2008-03-27 | 2008-09-10 | 武汉大学 | Construction method and application of two-color fluorescence report carrier |
| CN101505739A (en) * | 2006-03-30 | 2009-08-12 | Ptc医疗公司 | Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith |
| CN102121027A (en) * | 2010-09-30 | 2011-07-13 | 温州医学院 | Dual-fluorescence plasmid, dual-fluorescence plasmid-Based method for analyzing deoxyribonucleic acid (DNA) mismatch repair functional activity in living cell and application thereof |
| CN102839188A (en) * | 2012-10-14 | 2012-12-26 | 泰山医学院 | Homonymous tandem co-expression bi-color fluorescent protein report gene vector |
-
2013
- 2013-01-22 CN CN2013100233844A patent/CN103074346A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001038557A1 (en) * | 1999-11-24 | 2001-05-31 | The University Of New South Wales | Method of screening multiply transformed cells using bicistronic expression of fluorescent proteins |
| CN101505739A (en) * | 2006-03-30 | 2009-08-12 | Ptc医疗公司 | Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith |
| CN101260408A (en) * | 2008-03-27 | 2008-09-10 | 武汉大学 | Construction method and application of two-color fluorescence report carrier |
| CN102121027A (en) * | 2010-09-30 | 2011-07-13 | 温州医学院 | Dual-fluorescence plasmid, dual-fluorescence plasmid-Based method for analyzing deoxyribonucleic acid (DNA) mismatch repair functional activity in living cell and application thereof |
| CN102839188A (en) * | 2012-10-14 | 2012-12-26 | 泰山医学院 | Homonymous tandem co-expression bi-color fluorescent protein report gene vector |
Non-Patent Citations (2)
| Title |
|---|
| 尹涛等: "利用GFP/RFP双荧光指示载体鉴定特异性启动子功能", 《生物工程学报》, no. 12, 25 December 2008 (2008-12-25) * |
| 陈执中: "无义突变引起的遗传性疾病及其治疗新药的研究进展", 《食品与药品》, no. 01, 10 January 2008 (2008-01-10) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106979938A (en) * | 2017-03-09 | 2017-07-25 | 东华大学 | It is a kind of to carry out the method that destination gene expression is quantitatively detected using flow cytometer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering | |
| Rees et al. | Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery | |
| Hidalgo-Cantabrana et al. | Characterization and applications of Type I CRISPR-Cas systems | |
| KR20220004674A (en) | Methods and compositions for editing RNA | |
| Chen et al. | Adenine transversion editors enable precise, efficient A• T-to-C• G base editing in mammalian cells and embryos | |
| CN108148835A (en) | The sgRNA of CRISPR-Cas9 targeting knock out SLC30A1 genes and its specificity | |
| JP6959654B2 (en) | New minimum UTR sequence | |
| Trehan et al. | REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering | |
| Gaspar et al. | Improved minicircle DNA biosynthesis for gene therapy applications | |
| US20230212612A1 (en) | Genome editing system and method | |
| Chen et al. | Controlling the replication of a genomically recoded HIV-1 with a functional quadruplet codon in mammalian cells | |
| WO2023142594A1 (en) | Accurate pam-limitation-free adenine base editor and use thereof | |
| Wang et al. | CRISPR-Cas9 HDR system enhances AQP1 gene expression | |
| Jarczynska et al. | A versatile in vivo DNA assembly toolbox for fungal strain engineering | |
| Chen et al. | Engineering self-deliverable ribonucleoproteins for genome editing in the brain | |
| CN103074346A (en) | Method for establishing finite cell strain for screening read-though promoting drug | |
| Kumar et al. | DEAE-dextran transfection | |
| Thean et al. | To plate or to simply unfreeze, that is the question for optimal plasmid extraction | |
| Yao et al. | A direct RNA-to-RNA replication system for enhanced gene expression in bacteria | |
| CN110551722A (en) | Nucleic acid compound and preparation method and application thereof | |
| CN104789598A (en) | Method for constructing recombinant bombyx mori cypovirus for expressing red fluorescence protein | |
| Tao et al. | Cloning short DNA into plasmids by one‐step PCR | |
| Pan et al. | Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi’s sarcoma-associated herpesvirus ORF52 | |
| Shepherd et al. | Bioproduction of single-stranded DNA from isogenic miniphage | |
| Zhang et al. | Production of plasmid DNA encoding human hepatocyte growth factor for gene therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130501 |