CN101684478B - Method for constructing tandem expression small interfering RNA recombinant lentiviral vector - Google Patents
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Abstract
The invention discloses a method for constructing a tandem expression small interfering RNA recombinant lentiviral vector. The method comprises the following steps of: (1) constructing the siRNA recombinant lentiviral vector with single target spot: a, chemically synthesizing an oligonucleotide chain; b, annealing a sense chain and an anti-sense chain; and c, connecting an annealing fragment to the vector; (2) constructing the siRNA recombinant lentiviral vector with double target spots: a, amplifying a U6/H1-siRNA expression frame from the siRNA recombinant lentiviral vector with single target spot by utilizing the PCR; b, connecting the expression frame to the siRNA recombinant lentiviral vector with single target spot undergoing enzyme digestion by the Xhol; and c, performing the enzyme digestion, identification and screening of the clone forwardly inserted in the expression frame; and (3) constructing the siRNA recombinant lentiviral vector with multiple target points in tandem connection: inserting the U6/H1-siRNA expression frame in the siRNA recombinant lentiviral vector with double target spots to construct the siRNA recombinant lentiviral vector with triple target spots, and obtaining the siRNA recombinant lentiviral vector with multiple target points in tandem connection by repeating the step. The construction method is suitably used for the expression of the siRNA with multiple target genes as well as the research on the multiple target genes which are silent for a long time.
Description
Technical field
The present invention relates to biology field.Relate more specifically to a kind of construction process of tandem expression small interfering RNA recombinant lentiviral vector.
Background technology
(RNA interference RNAi) is meant sequence-specific PTGS (post-transcriptional gene silencing, process PTGS) that is triggered by double-stranded RNA in the RNA interference.At present; RNA interferential mechanism is illustrated basically: double-stranded RNA endogenous or external source is cut into the siRNA (siRNA) with 21～23nt of the outstanding 3 ' end of 2 bases by Dicer in tenuigenin; (RNA-induced silencing complex RISC) combines for the latter and RNA inductive silencing complex.SiRNA is unwind in RISC, and wherein antisense strand guides RISC target complementary mRNA with it, and RISC is cutting mRNA in the middle of complementary sequence then, thereby suppresses target gene expression.
Although find so far only 11 years from the RNAi phenomenon, RNAi has been widely used in Basic of Biology research and Application Areas as a kind of gene silencing instrument.5 kinds of methods that prepare siRNA comparatively commonly used at present comprise: 1) chemosynthesis; 2) in-vitro transcription; 3) the disconnected dsRNAs of lengthy motion picture is through RNase III enzyme liberating; 4) the siRNA expression cassette of PCR preparation is expressed in cell; 5) the siRNA expression vector is expressed siRNA in cell.Above method all has relative merits separately: the siRNAs of preceding 4 kinds of methods preparation or siRNA expression cassette are gone into cell through transfection or electricity transduction, can realize that transient gene is reticent, and be then not too suitable for the albumen that the transformation period is long.The siRNA expression vector then can utilize the antibiotic marker on the carrier to set up metastable long-term gene silencing cell strain, continues to suppress target gene expression.Most siRNA expression vectors utilizes III type rna polymerase promoter to handle siRNA and in mammalian cell, expresses, and the III type promotor that adopts usually comprises people source and mouse source U6 promotor and people source H1 promotor.At present, a lot of biotech firms have developed the siRNA expression vector based on U6 or H1 promotor, and representative product comprises: the pSilencer series of Ambion company; The siLentGene series of Promega company; The BLOCK-iT series of Invitrogen company, etc.
The siRNA expression vector can be divided into two types of plasmid vector and virus vector.There is the low shortcoming of transfection efficiency for some difficult cells transfected plasmid vector; So beginning to research and develop virus vector, many in recent years biotech firms express siRNA; It is advantageous that directly the high-level efficiency cells infected carries out the research of gene silencing, avoid owing to the low inconvenience that brings of plasmid transfection efficient.Virus vector commonly used at present comprises: retroviral vector, adenovirus carrier, lentiviral vectors.Wherein lentiviral vectors has such as infecting division and non-division stage cell, can be integrated in advantage such as host genome and quilt application more and more widely.Representative siRNA lentiviral vectors such as pLentiLox3.7, this carrier contain one and handle siRNA by mouse source U6 promotor and express.
Yet; That has developed at present only is suitable for the expression of the siRNA of single target spot based on the siRNA slow virus expression vector of III type promotor; And increasing fundamental research and applied research need reticent a plurality of target genes simultaneously; In order to address this problem, the present invention has designed the construction process of the lentiviral vectors of a kind of tandem expression siRNA, is applicable to the experimental study and the applied research of a plurality of target genes of long-term silence.
Summary of the invention
The objective of the invention is to be to provide a kind of construction process of tandem expression siRNA recombined lentivirus vector; Overcome the limitation that existing siRNA expression vector only is applicable to the siRNA that expresses single target spot; Can express siRNA simultaneously to a plurality of target genes; And be suitable for the foundation of stable cell lines, thereby be applicable to the experimental study and the applied research of a plurality of target genes of long-term silence.
The construction process of this tandem expression siRNA recombined lentivirus vector may further comprise the steps:
1, the promoter engineering of lentiviral vectors:
The lentiviral vectors that the applicant transforms is pLentiLox3.7 (Rubinson and Dillon, NatureGenetics, 2003).This carrier contains the expression that a mouse source U6 promotor is used for controlling siRNA.Promotor for fear of series connection siRNA is single and cause that homologous recombination causes losing of siRNA expression cassette; The applicant replaces the mouse source U6 promotor of this carrier respectively and is people source U6 promotor and people source H1 promotor, and improved lentiviral vectors is called after pLLU6, pLLH1 (Escherichia coli Stbl3/pLLU6 CCTCCM 209135 respectively; Escherichia coli Stbl3/pLLH1 CCTCC M 209136, preservation date: on June 25th, 2009, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University)
2, the construction process of tandem expression siRNA recombined lentivirus vector
2.1 the structure of the siRNA recombined lentivirus vector of single target spot
2.1.1 the chemical synthetic oligonucleotide chain, form is following:
Sense strand: 5 '-(19N)-(TTCAAGAGA)-(N91)-TTTTTTC-3 '
Antisense strand: complementary with sense strand, and add at 5 ' end and to go up TCGA to produce Xho1 viscosity protruding terminus
Annotate: 19N representes 19 base sequences of siRNA target spot, and N19 representes the reverse complementary sequence of 19N;
2.1.2 sense strand, antisense strand annealing form the terminal fragment of band Xho1 sticky end peace;
2.1.3 above fragment is connected to carrier pLLU6 or pLLH1 (carrier is by KspA1 and Xho1 double digestion);
2.1.4 transformed into escherichia coli Stbl3 competent cell;
2.1.5 screening positive clone, and order-checking is identified;
2.2 the structure of the siRNA recombined lentivirus vector of two target spots
2.2.1 utilize PCR " U6/H1-siRNA " expression cassette that from the siRNA recombined lentivirus vector of single target spot, increases, and make it have the Xho1 sticky end with Sal1, Xho1 double digestion
For " U6-siRNA " expression cassette, the PCR primer sequence is following:
Upstream primer: U6-siR-1 (5 '-TAACGC
GTCGACCCCCAGTGGAAAGACGCGCA-3 ')
Sal1
Downstream primer: U6-siR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
For " H1-siRNA " expression cassette, the PCR primer sequence is following:
Upstream primer: H 1-siR-1 (5 '-TAACGC
GTCGACAATTCATATTTGCATGTCGC-3 ')
Sal1
Downstream primer: H1-siR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
2.2.2 " U6/H1-siRNA " expression cassette is connected to the siRNA recombined lentivirus vector of single target spot of cutting through the Xhol enzyme, and transformed into escherichia coli Stbl3 competent cell, bacterium colony PCR screening positive clone;
2.2.3 enzyme is cut the clone of evaluation and screening " insertion of fragment forward "
Because there is the possibility of positive and negative both direction in the segmental insertion of " U6/H1-siRNA " expression cassette, in order to be fit to the structure and the Screening and Identification of series connection siRNA recombined lentivirus vector, this step is only selected the clone that forward inserts.Concrete authentication method is: with Xba1, Xho1 double digestion clone to be measured, establish and do not insert segmental carrier for contrast, if the endonuclease bamhi size greater than contrast, then is " forward insertion "; If the endonuclease bamhi size is identical with contrast, then be " the reverse insertion ";
2.2.4 order-checking is identified
2.3 the structure of the series connection siRNA recombined lentivirus vector of many target spots
" U6/H1-siRNA " expression cassette is inserted into the siRNA recombined lentivirus vector of two target spots, promptly constructs the siRNA recombined lentivirus vector of three target spots,, can obtain the placed in-line siRNA recombined lentivirus vector of many target spots with this construction strategy.
In the application example of one group of interference virus of AIDS; The applicant has made up the series connection siRNA recombined lentivirus vector to 3 target spots; Experimental result confirms this lentiviral vectors ability works better, and placed in-line siRNA expression cassette is (referring to the embodiment 3) that works alone.
The invention has the beneficial effects as follows: the lentiviral vectors of (1) tandem expression siRNA provided by the present invention is applicable to reticent a plurality of target genes simultaneously; Be suitable for having the fundamental research and the applied research of demand like this; Such as: in the treating AIDS field; Different target spots with siRNA while a plurality of virogenes of target or the same virogene of target can effectively prevent or delay viral escape; (2) the present invention is suitable for the foundation of stable cell lines, thereby can reticent steadily in the long term target gene; (3) be easy to realize the directed insertion of siRNA expression cassette; (4) lentiviral vectors of tandem expression siRNA provided by the invention provides U6, two kinds of promotors of H1 selective, can avoid to a certain extent causing losing of siRNA expression cassette because of the homologous recombination that repeatedly repeats to cause with a kind of promotor.
Description of drawings
Fig. 1 is the promoter engineering synoptic diagram of lentiviral vectors.Use people source U6 and people source H1 promotor to replace the mouse source U6 promotor on the pLentiLox 3.7 respectively, obtain pLLU6, pLLH1 carrier from the pSilencer plasmid.
Fig. 2 is the structure schema of tandem expression small interfering RNA recombinant lentiviral vector.At first, the oligonucleotide fragment of the coding siRNA of chemosynthesis is connected to pLLU6, pLLH1 carrier, obtains the siRNA recombined lentivirus vector of single target spot through KspA1 and Xho1 restriction enzyme site; Then, pcr amplification " U6/H1-siRNA " expression cassette is connected to the siRNA recombined lentivirus vector of single target spot that the Xho1 enzyme cuts, thereby obtains the siRNA recombined lentivirus vector of two target spots; At last, construction of strategy goes out the recombined lentivirus vector of tandem expression siRNA according to this.
Fig. 3 is the lentiviral vectors application example of tandem expression small interfering RNA.This instance is chosen the required host cell membrane PROTEIN C CR5 of hiv gene rev, pol, tat and virus infection cell as target spot, constructs the siRNA recombined lentivirus vector of single target spot, two target spot, three target spots.This sample result shows: the lentiviral vectors ability works better of tandem expression small interfering RNA provided by the invention, and placed in-line siRNA expression cassette works alone.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further specified.
Embodiment 1: the promoter engineering of lentiviral vectors
The lentiviral vectors that the applicant transforms is pLentiLox3.7 (Rubinson and Dillon, NatureGenetics, 2003).This carrier contains the expression that a mouse source U6 promotor is used for controlling siRNA, also contains reporter gene EGFP simultaneously and is convenient to selected by flow cytometry apoptosis.Promotor for fear of series connection siRNA is single and cause that homologous recombination causes losing of siRNA expression cassette; The applicant replaces the mouse source U6 promotor of this carrier respectively and is people source U6 promotor and people source H1 promotor (people source U6 promotor and people source H1 promotor obtain through PCR from Ambion Company products pSilencer2.0_U6 and pSilencer3.0_H1), and improved lentiviral vectors is called after pLLU6, pLLH1 (Escherichia coli Stbl3/pLLU6CCTCC M209135 respectively; Escherichia coli Stbl3/pLLH1 CCTCC M 209136).The promoter engineering synoptic diagram is seen shown in Figure 1.Concrete transformation process is following:
1.1PCR amplification U6 and H1 promoter fragment;
For the U6 promotor, the PCR primer is following:
Upstream primer: U6-1 (5 '-ACA
TCTAGACCCCAGTGGAAAGACGCGCAG-3 ')
Xba1
Downstream primer: U6-2 (5 '-ACG
GTTAACGATCCCGCGTCCTTTCCACAA-3 ')
KspA1
For the H1 promotor, the PCR primer is following:
Upstream primer: H1-1 (5 '-CGG
TCTAGAAATTCATATTTGCATGTCGCTA-3 ')
Xba1
Downstream primer: H1-2 (5 '-AGC
GTTAACCGAGTGGTCTCATACAGAACTT-3 ')
KspA1
1.2 above promoter fragment is cloned into pEGM-T carrier (available from Promega company) with T-A clone strategy;
1.3, isolate the U6/H1 promoter fragment of band sticky end with Xba1 and the above reorganization of KspA1 double digestion pEGM-T carrier;
1.4, obtain carrier with corresponding sticky end with Xba1 and KspA1 double digestion pLentiLox 3.7;
1.5 the U6/H1 promoter fragment is connected to above carrier, obtain the lentiviral vectors of promoter engineering, respectively called after pLLU6, pLLH 1 (Escherichia coli Stbl3/pLLU6CCTCC M 209135; Escherichia coli Stbl3/pLLH1 CCTCC M 209136).
Embodiment 2: the construction process of tandem expression siRNA recombined lentivirus vector
2.1 the structure of the siRNA recombined lentivirus vector of single target spot (referring to Fig. 2 step I)
2.1.1 the chemical synthetic oligonucleotide chain, form is following:
Sense strand: 5 '-(19N)-(TTCAAGAGA)-(N91)-TTTTTTC-3 '
Antisense strand: complementary with sense strand, and add at 5 ' end and to go up TCGA to produce Xho1 viscosity protruding terminus
Annotate: 1) 19N representes 19 base sequences of siRNA target spot, and N19 representes the reverse complementary sequence of 19N;
2) (TCAAGAGA) instruct to generate " ring " zone of siRNA precursor shRNA (bobby pin RNA),
This sequence is selected from (Brummelkamp et al., Science, 2002)
2.1.2 sense strand, antisense strand annealing form the terminal fragment of band Xho1 sticky end peace;
2.1.3 above fragment is connected to carrier pLLU6 or pLLH1 (carrier is by KspA1 and Xho1 double digestion);
2.1.4 transformed into escherichia coli Stbl3 competent cell (the Stbl3 bacterial strain is available from Invitrogen company);
2.1.5 screening positive clone, and order-checking is identified;
2.2 the structure of the siRNA recombined lentivirus vector of two target spots (referring to Fig. 2 Step II)
2.2.1 utilize PCR " U6/H1-siRNA " expression cassette that from the siRNA recombined lentivirus vector of single target spot, increases, and make it have the Xho1 sticky end with Sal1, Xho1 double digestion
For " U6-siRNA " expression cassette, the PCR primer sequence is following:
Upstream primer: U6-siR-1 (5 '-TAACGC
GTCGACCCCCAGTGGAAAGACGCGCA-3 ')
Sal1
Downstream primer: U6-siR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
For " H1-siRNA " expression cassette, the PCR primer sequence is following:
Upstream primer: H 1-siR-1 (5 '-TAACGC
GTCGACAATTCATATTTGCATGTCGC-3 ')
Sal1
Downstream primer: H1-siR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
2.2.2 " U6/H1-siRNA " expression cassette is connected to the siRNA recombined lentivirus vector of single target spot of cutting through the Xhol enzyme, and transformed into escherichia coli Stbl3 competent cell, bacterium colony PCR screening positive clone;
2.2.3 enzyme is cut the clone of evaluation and screening " insertion of fragment forward "
Because there is the possibility (is forward with closure shown in Fig. 2 Step II) of positive and negative both direction in the segmental insertion of " U6/H1-siRNA " expression cassette; In order to be fit to the structure and the Screening and Identification of series connection siRNA recombined lentivirus vector, this step is only selected the clone that forward inserts.Concrete authentication method is: with Xba1, Xho1 double digestion clone to be measured, establish and do not insert segmental carrier for contrast, if the endonuclease bamhi size greater than contrast, then is " forward insertion "; If the endonuclease bamhi size is identical with contrast, then be " the reverse insertion ";
2.2.4 order-checking is identified
Sequencing primer: 5 '-CAGTGCAGGGGAAAGAATAGTAGAC-3 '
(Rubinson?and?Dillon，Nature?Genetics，2003)；
2.3 the structure of the series connection siRNA recombined lentivirus vector of many target spots (referring to Fig. 2 Step II I)
" U6/H 1-siRNA " expression cassette is inserted into the siRNA recombined lentivirus vector of two target spots, promptly constructs the siRNA recombined lentivirus vector of three target spots,, can obtain the placed in-line siRNA recombined lentivirus vector of many target spots with this construction strategy.
Embodiment 3: the lentiviral vectors application example of tandem expression siRNA
Whether can works better for the lentiviral vectors of verifying tandem expression siRNA, the applicant explains with the application example that disturbs virus of AIDS:
In this embodiment; The applicant chooses the required host cell membrane PROTEIN C CR5 of hiv gene rev, pol, tat and virus infection cell as target spot, utilizes the lentiviral vectors of tandem expression siRNA provided by the present invention to construct the siRNA recombined lentivirus vector of single target spot, two target spot, three target spots.SiRNA expression cassette array mode is as shown in the table:
| Single target spot | H1?CCR5 U6?rev U6?pol U6?tat |
| Two target spots | H1?CCR5_U6?rev H1?CCR5_U6?pol H1?CCR5_U6?tat |
| Three target spots | H1?CCR5_U6?rev_U6?pol H1?CCR5_U6?rev_U6?tat H1?CCR5_U6?poL_U6?tat |
The applicant with above siRNA recombined lentivirus vector respectively with pNL4-3.Luc.R-E-(Landau NR; Viology; 1995) cotransfection HEK-293T cell; Detect the plain enzymic activity of relative fluorescence after 24 hours, the result is (annotate: pNL4-3.Luc.R-E-is the HIV replicon of band luciferase reporter gene, and its levels of replication can be represented by uciferase activity) shown in Fig. 3 (A).The result shows: each siRNA recombined lentivirus vector is to the restraining effect that all has in various degree of duplicating of HIV; Wherein the effect of Combination of rev-pol, rev-tat, pol-tat siRNA is higher than its effect of performance separately separately; Explain that rev, pol, tatsiRNA expression cassette can work alone, and action effect has synergistic effect after making up in twos.The concrete outcome analytical data is as shown in the table:
(annotate: the CCR5siRNA expression cassette does not play a role in above-mentioned experiment, because used HEK-293T cell is not expressed the CCR5 membranin, and pNL4-3.Luc.R-E-is that liposome transfection gets into cell, need not membrane receptor CCR5 mediation)
In order to verify can working alone of CCR5siRNA expression cassette; The applicant utilizes constructed siRNA recombined lentivirus vector pLLH1 CCR5 and pLLH1 CCR5_U6 rev_U6 tat to set up stable cell lines (the KewalRamani V and Littman DR based on GhostCD4/CCR5; J Virol, 1999) (this clone can stably express membranin CCR5).The CCR5 antibody (available from eBioscience company) of using the PE mark has then carried out streaming detection (result is shown in Fig. 3 (B), and gray area is a sample, the negative contrast of hollow area) to the stable cell lines of above foundation.The result shows: by the equal effective down-regulation of CCR 5 expression level of the CCR5siRNA of pLLH1 CCR5 and pLLH1 CCR5_U6rev_U6 tat expression; And the two no significant difference (reducing 77.5% and 79.9% respectively), thereby explanation: the working alone of the CCR5siRNA expression cassette in the siRNA recombined lentivirus vector of tandem expression.
In sum, the application example explanation that present embodiment relates to: the lentiviral vectors ability works better of tandem expression siRNA provided by the invention, and placed in-line siRNA expression cassette works alone.
Embodiment 4: the packing of the slow virus of tandem expression siRNA.
4.1 transfection preceding 24 hours shops HEK-293T cell (available from CCTCC) is in the Tissue Culture Dish of a diameter 7.5cm;
4.2 cell degree of converging is advisable with 40～60% during transfection, operates following plasmid co-transfection cell with the calcium phosphate transfection of standard:
4 μ g siRNA recombined lentivirus vectors
2μg?pLP1
2μg?pLP2
2μg?pVSVG
(annotate: pLP1, pLP2, pVSVG are slow virus auxiliary package plasmids, available from Invitrogen company); 4.3 collected viral supernatant (being cell culture medium) respectively in 24 hours, 48 hours, 72 hours after the transfection, and supply substratum; Mix the viral supernatant of collecting for 3 times afterwards;
4.4 virus titer detects: with viral supernatant with 10 times of gradient dilutions; Infect the HEK-293T cell respectively and (add polybrene (available from SIGMA company) in the substratum; Final concentration is 8 μ g/ml); Streaming detects GFP positive cell ratio after 48 hours, and calculates virus titer with the extent of dilution of ratio between 0.1-10%.
Embodiment 5: utilize the slow virus of tandem expression siRNA to set up stable cell lines.
5.1 infect Ghost CD4/CCR5 cell with gradient m.o.i. with the slow virus of being packed among the embodiment 4; Infect streaming detection GFP positive cell ratio after 48 hours; Select its ratio 10% following person and carry out aseptic airflow classification (annotate: opt ratio 10% following person why is to be infected again by slow virus and influence the accuracy of subsequent experimental for fear of cell);
Cell to the cell quantity of cultivating sorting 5.2 go down to posterity is suitable;
5.3 streaming detects GFP positive cell ratio once more, to confirm the purity of stable cell lines.
SEQUENCE?LISTING
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Claims (3)
1. the lentiviral vectors pLLU6 of a transformation, this carrier is Escherichia coli StbI3/pLLU6, preserving number is CCTCC M 209135.
2. the lentiviral vectors pLLH1 of a transformation, this carrier is Escherichia coli StbI3/pLLH1, preserving number is CCTCC M 209136.
3. the construction process of a tandem expression siRNA recombined lentivirus vector may further comprise the steps:
(1) structure of the siRNA recombined lentivirus vector of single target spot:
A, chemical synthetic oligonucleotide chain, form is following:
Sense strand: 5 '-(19N)-(TTCAAGAGA)-(N91)-TTTTTTC-3 '
Antisense strand: complementary with sense strand, go up TCGA to produce Xho1 viscosity protruding terminus in 5 ' end interpolation;
Described 19N representes 19 base sequences of siRNA target spot, and N19 representes the reverse complementary sequence of 19N;
B, sense strand, antisense strand annealing form the terminal fragment of band Xho1 sticky end peace;
C, above fragment is connected to described carrier pLLU6 of claim 1 or the described carrier pLLH1 of claim 2;
D, transformed into escherichia coli StbI3 competent cell;
E, screening positive clone, and order-checking is identified;
(2) structure of the siRNA recombined lentivirus vector of two target spots:
A, utilize the PCR U6/H1-siRNA expression cassette that from the siRNA recombined lentivirus vector of single target spot, increases, and make it have the Xho1 sticky end with Sal1, Xho1 double digestion
For the U6-siRNA expression cassette, the PCR primer sequence is following:
Upstream primer: U6-siR-1 (5 '-TAACGC
GTCGACCCCCAGTGGAAAGACGCGCA-3 ')
Sal1
Downstream primer: U6-siR-2 (5 '-ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
For the H1-siRNA expression cassette, the PCR primer sequence is following:
Upstream primer: H1-siR-1 (5 '-TAACGC
GTCGACAATTCATATTTGCATGTCGC-3 ')
Sal1
Downstream primer: H1-siR-2 (5 ' ATACCG
CTCGAGCTCGTGAAGCGAGCTTATCGATACC-3 ')
Xho1
B, the U6/H1-siRNA expression cassette is connected to the siRNA recombined lentivirus vector of single target spot of cutting through the XhoI enzyme, and transformed into escherichia coli StbI3 competent cell, bacterium colony PCR screening positive clone;
C, enzyme are cut the clone that evaluation and screening fragment forward inserts; This step is only selected the clone that forward inserts, and concrete authentication method is: with Xba1, Xho1 double digestion clone to be measured, establish and do not insert segmental carrier for contrasting; The endonuclease bamhi size is greater than contrast; For forward inserts, the endonuclease bamhi size is identical with contrast, is reverse insertion;
D, order-checking are identified;
(3) structure of the series connection siRNA recombined lentivirus vector of many target spots:
The U6/H1-siRNA expression cassette is inserted into the siRNA recombined lentivirus vector of two target spots, promptly constructs the siRNA recombined lentivirus vector of three target spots,, can obtain the placed in-line siRNA recombined lentivirus vector of many target spots with this construction strategy.
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| CN104805085A (en) * | 2014-01-29 | 2015-07-29 | 江苏命码生物科技有限公司 | Tandem expressed siRNA and use of tandem expressed siRNA in treatment on chronic lymphocytic leukemia |
| CN103834692B (en) * | 2014-02-27 | 2016-03-30 | 南京医科大学 | A kind of lentiviral vectors for expressing lncRNA and application thereof |
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| WO2004022722A3 (en) * | 2002-09-06 | 2004-11-25 | Massachusetts Inst Technology | Lentiviral vectors, related reagents, and methods of use thereof |
| CN1738648A (en) * | 2003-01-17 | 2006-02-22 | 佛罗里达大学研究基金会有限公司 | Small interference RNA gene therapy |
| CN101113169A (en) * | 2006-07-28 | 2008-01-30 | 中国科学院广州分院 | Use of sorting protein SNX10 for suppressing growth of tumour cell |
| CN101184852A (en) * | 2005-03-09 | 2008-05-21 | 贝勒医学院 | Direct reversal of the suppressive function of cd4+ regulatory t cells via toll-like receptor 8 signaling |
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2009
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004022722A3 (en) * | 2002-09-06 | 2004-11-25 | Massachusetts Inst Technology | Lentiviral vectors, related reagents, and methods of use thereof |
| CN1738648A (en) * | 2003-01-17 | 2006-02-22 | 佛罗里达大学研究基金会有限公司 | Small interference RNA gene therapy |
| CN101184852A (en) * | 2005-03-09 | 2008-05-21 | 贝勒医学院 | Direct reversal of the suppressive function of cd4+ regulatory t cells via toll-like receptor 8 signaling |
| CN101113169A (en) * | 2006-07-28 | 2008-01-30 | 中国科学院广州分院 | Use of sorting protein SNX10 for suppressing growth of tumour cell |
Non-Patent Citations (3)
| Title |
|---|
| 叶雪石.慢病毒介导的RNA干扰逆转单因素耐药细胞系K562/MDR1耐药性的研究.《四川大学临床医学博士专业学位论文》.2008,全文. * |
| 李文刚 等.腺病毒介导反义RNA抑制HIV-1辅助受体CCR5和CXCR4表达.《中国医学科学院学报》.2006,第28卷(第5期),626-631. * |
| 郭德银.RNA干扰在病毒研究和控制中的应用.《第二届中国青年学者微生物遗传学学术研讨会》.2006,176. * |
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