CN103834657B - A kind of method of artificial DNA molecule and expression target gene - Google Patents
A kind of method of artificial DNA molecule and expression target gene Download PDFInfo
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Abstract
The invention discloses a kind of method of artificial DNA molecule and expression target gene.Does is the base sequence of this artificial DNA molecule SEQ ID NO.2, can encode TALE-TF1, can described TALE-TF1 identify SEQ ID 28-48 position in NO.1; Or the base sequence of described artificial DNA molecule is SEQ ID NO.3, can encode TALE-TF2, can described TALE-TF2 identify SEQ ID 42-62 position in NO.1; Or the base sequence of described artificial DNA molecule is SEQ ID NO.4, can encode TALE-TF3, can described TALE-TF3 identify SEQ ID 56-76 position in NO.1.Artificial DNA molecule of the present invention can activate beta-casein gene promotor in Sheep Fibroblast, and the structure for Sheep Mammary bio-reactor provides the detection technique of direct convenience.
Description
Technical field
The present invention relates to gene engineering field, particularly relate to a kind of artificial DNA molecule, and utilize this artificial DNA molecule to express the method for target gene.
Background technology
Sheep beta-casein gene high expression level in mammary gland, also can express in skin and poisonous T lymphocyte (cytotoxicTlymphocytes).Casein is the main protein component in sheep Ruzhong, is divided into 4 classes: α according to structure
s1, α
s2, β and κ.In mammary tissue, 4 kinds of casein gene mRNA translation efficiencies are different, α
s1α with the mRNA translation efficiency of β
s2with 3 to 4 times of κ.In sum, beta-casein gene site is the appropriate targets preparing galactophore biological reactor.
Whether effective for confirming the expression cassette of beta-casein gene promoters driven, Kolb etc. (1999) adopt mammary epithelial cell.Domestic galactophore biological reactor research aspect, also adopts the validity of mammary epithelial cell checking expression cassette.If beta-casein gene promotor can be activated in Sheep Fibroblast, detect the expression of results of recombinant expressed frame, the structure for Sheep Mammary bio-reactor is provided the detection technique of direct convenience.
Summary of the invention
The object of the invention is to propose a kind of artificial DNA molecule, and utilize this artificial DNA molecule to express the method for target gene.
In the present invention, term " TALE " refers to transcriptional activation sample effector.
In the present invention, term " TALE-TFs " refers to TALE transcription factor, i.e. TALEtranscriptionfactors.
In order to solve the problems of the technologies described above, present invention employs following technical scheme:
Can be encoded the artificial DNA molecule of TALE-TFs, and the base sequence of described artificial DNA molecule is SEQIDNO.2, and can encode TALE-TF1, and described TALE-TF1 can identify the 28-48 position in SEQIDNO.1; Or the base sequence of described artificial DNA molecule is SEQIDNO.3, can encode TALE-TF2, and described TALE-TF2 can identify the 42-62 position in SEQIDNO.1; Or the base sequence of described artificial DNA molecule is SEQIDNO.4, can encode TALE-TF3, and described TALE-TF3 can identify the 56-76 position in SEQIDNO.1.
The present invention also provides a kind of expression vector, and described expression vector comprises artificial DNA molecule according to claim 1, and it can express TALE-TF1, TALE-TF2 or TALE-TF3.
The present invention also provides a kind of reconstitution cell, and described reconstitution cell uses the prokaryotic cell prokaryocyte or eukaryotic cell that described in claim 2, expression vector transforms.
Express a method for target gene, comprise the following steps:
Steps A, target gene is proceeded to sheep beta-casein gene, build the sheep beta-casein gene expression vector containing target gene;
Step B, according to sheep beta-casein gene promotor target sequence design TALE arrangement, and the expression vector of construction expression TALE-TFs; Wherein said sheep beta-casein gene promotor target sequence is the 28-48 position in SEQIDNO.1, or target sequence is the 42-62 position in SEQIDNO.1, or target sequence is the 56-76 position in SEQIDNO.1;
Step C, obtain the Sheep Fibroblast after transfection with the expression vector cotransfection Sheep Fibroblast of described sheep beta-casein gene expression vector containing target gene and described expression TALE-TFs.
Further, the expression vector of expressing TALE-TFs described in step B comprises SEQIDNO.2 base sequence, or SEQIDNO.3 base sequence, or SEQIDNO.4 base sequence.
Whether compared with prior art, beneficial effect of the present invention is: effective for confirming the expression cassette of restructuring, all adopts both at home and abroad and detects in mammary epithelial cell.And utilize TALE-TFs technology to enable the Sheep Fibroblast of not expressing beta-casein gene express beta-casein gene, also beta-casein gene---the external source integrator gene expression cassette activating restructuring with TALE-TFs in Sheep Fibroblast can just be realized, a kind of convenient detection approach can be provided, without the need to cultivating mammary epithelial cell for the expression cassette of the beta-casein promoters driven of vitro detection restructuring.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is sheep beta-casein gene structural representation;
Fig. 2 is pTY-ShE1LB-2BsmBI-ShE1RB carrier structure schematic diagram;
Fig. 3 is pTY-ShE2LB-2BsmBI-ShE2RB carrier structure schematic diagram;
Fig. 4 is pShE1lB-Red-pA-ShE1RB reporter gene expression carrier structure schematic diagram;
Fig. 5 is pShE2LB-Red-pA-ShE2RB reporter gene expression carrier structure schematic diagram;
Fig. 6 is TALE-TFs and Red reporter plasmid cotransfection sheep embryo inoblast green fluorescent reporter genetic expression figure;
Fig. 7 is TALE-TFs and Red reporter plasmid cotransfection sheep embryo inoblast red fluorescence reporter gene expression figure;
Fig. 8 is that TALE-TFs induces sheep embryo inoblast beta-casein gene to express reverse transcription PCR detection figure;
Fig. 9 is that TALE-TFs induces sheep embryo inoblast beta-casein gene to express quantitative fluorescent PCR analysis chart.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, but not as a limitation of the invention.
Embodiment 1 vector construction
The primer used in vector construction step in embodiment 1 lists in table 1.
Table 1 Primer and sequence
| Title | Sequence |
| ShE1F | GCTTTTGCAAATGGGCTCCT |
| ShE2R | GCCACATTAGCTTAAAGAACACTGG |
| TYShE1LBF | GCTACGCTCGAGCCTTCCCTCATTTGCCCTTCCA |
| TYGDShE1R | CTAGCAAGCTTCTGTACGTCTCTGGCTCTCGATTCCTTTTTTCCAAAGTAGAGGAGAAG |
| ShE2-Oli1 | AGCCATGAAGGTCCTCATCCTTGCCTGTCTGGTGGCTCTGGCCCTTGCAGAGACGAGA |
| ShE2-Oli2 | AGCTTCTCGTCTCTGCAAGGGCCAGAGCCACCAGACAGGCAAGGATGAGGACCTTCAT |
| TYShE1RBF | CTCTTCAAGCTTCCTATTCGTCTCCAGAGAATCTCAGATATAATTTCAGTGTATCT |
| TYShE1RBR | TGCCTACAATTGTACCAGGGTGTCAGTTTCTCTT |
| TYShE2LBR | GACAGTAAGCTTTCGTCTCTGCAAGGGCCAGAGCCACCAGACAGGCA |
| TYShE2RBF | CTCTTCAAGCTTCAGTTCCGTCTCCAGAGAGGTAAATACAGAAAAAAATGTTGA |
| TYShE2RBR | TCAGTTCTTAAGCTGCTGCTGCTGCTAAGTCACTT |
| TYShpAF | CTCTTCAAGCTTCCTATTCTGATTCGTCTCGCGGCCGCGACTCTAGATCATAATCA |
| TYShRedF | TCGTACCGTCTCCTTGCAATGGCCTCCTCCGAGAACGTCAT |
| TYShRedR | GTCAGTCGTCTCCCTCTGTTTGGACAAACCACAACTAGAATGCA |
| ShGAPDHRTF | TCGGAGTGAACGGATTTGGC |
| ShGAPDHRTR | TGATGACGAGCTTCCCGTTC |
| ShbetaRTF | AGAACTCAATGTAGTCGGTGAGAC |
| ShbetaRTR | GTTAGGGATGGGCCCAGTGAA |
Take ovine genome as masterplate, ShE1F and ShE2R is primer, and pcr amplification obtains 4580bpDNA fragment 1, as shown in Figure 1, is sheep beta-casein gene structural representation, ↓ represent ShE1F primer sites; ↑ represent ShE2R primer sites; That DNA fragmentation 1, E1, E2, E3, E4, E5, E6, E7, E8, E9 represents the 1st to the 9th exon respectively between primer pair.With sheep beta-casein gene TATAbox for benchmark, amplified fragments comprises TATAbox upstream 1065bp5 ' promoter sequence, and downstream 3504bp is to the 4th intron partial sequence.
With DNA fragmentation 1 for masterplate, increase with primer TYShE1LBF/TYGDShE1LBR and TYShE1RBF/TYShE1RBR, obtain homologous recombination left arm DNA fragmentation 2 and the right arm DNA fragmentation 3 of sheep β casein gene First Exon target spot.Adopt XhoI and HindIII double digestion fragment 2 and pDS-Red2-1 plasmid (buying from ClontechLaboratories, INC.), T4 ligase enzyme connects, transformation of E. coli DH5a competent cell, obtains excessive vector pDS-Red-GDLB.In order to signal peptide complete for sheep β casein gene is stitched together, ShE2-Oli1/ShE2-Oli2(Invitrogen Beijing Company is synthesized) annealing renaturation, obtain the DNA fragmentation containing 4 base cohesive ends.Cut pDS-Red-GDLB carrier with HindIII/BsmBI enzyme, glue reclaims DNA fragmentation, and this fragment is annealed renaturation fragment through the connection of T4DNA ligase enzyme with ShE2-Oli1/ShE2-Oli2, obtains pDS-Red-ShE1LB carrier.Application HindIII/MnuI double digestion right arm DNA fragmentation 3 and pDS-RED-ShE1LB carrier, glue reclaims DNA fragmentation, T4DNA ligase enzyme connects homologous segment, obtains pTY-ShE1LB-2BsmBI-ShE1RB, pTY-ShE1LB-2BsmBI-ShE1RB carrier structure schematic diagram as shown in Figure 2.
With DNA fragmentation I for masterplate, increase with primer TYShE1LBF/TYShE2LBR and TYShE2RBF/TYShE2RBR, obtain homologous recombination left arm DNA fragmentation 4 and the right arm DNA fragmentation 5 of sheep β casein gene Second Exon target spot.Adopt XhoI and HindIII double digestion fragment 4 and pDS-Red2-N1 plasmid, glue reclaims DNA fragmentation, and T4DNA ligase enzyme connects, transformation of E. coli DH5a competent cell, obtains carrier pDS-Red-ShE2LB.Application HindIII/AflII double digestion right arm DNA fragmentation 3 and pDS-RED-ShE2LB carrier, glue reclaims DNA fragmentation, and T4DNA ligase enzyme connects homologous segment, obtains carrier pTY-ShE2LB-2BsmBI-ShE2RB.PTY-ShE2LB-2BsmBI-ShE2RB carrier structure schematic diagram as shown in Figure 3.
Masterplate adopts pDS-Red2-N1 plasmid, and TYShRedF/TYShRedR primer PCR increases, and obtains red fluorescent protein Red+polyA gene fragment.BsmBI enzyme cuts Red+polyA gene fragment, pTY-ShE1LB-2BsmBI-ShE1RB carrier and pTY-ShE2LB-2BsmBI-ShE2RB carrier.Glue reclaims corresponding gene fragment.T4DNA ligase enzyme connects Red+polyA gene fragment and pTY-ShE1LB-2BsmBI-ShE1RB carrier, and obtain pShE1lB-Red-pA-ShE1RB reporter gene expression carrier, pShE1lB-Red-pA-ShE1RB reporter gene expression carrier structure as shown in Figure 4.T4DNA ligase enzyme connects Red+polyA gene fragment and pTY-ShE2LB-2BsmBI-ShE2RB carrier, and obtain pShE2LB-Red-pA-ShE2RB reporter gene expression carrier, pShE2LB-Red-pA-ShE2RB reporter gene expression carrier structure as shown in Figure 5.
In vector construction process, relate to pcr amplified dna fragment, all adopt the high-fidelity of TAKARA company
maxDNAPolymerase(R050A), pcr amplification program: 98 DEG C of 10s; 55 DEG C of 10s; 72 DEG C of 10s; 35 circulations, reaction system is in table 2.
Table 2PCR amplification of DNA fragments
In vector construction process, relate to connection DNA fragmentation, all adopt the T4DNA ligase enzyme (2011A) of TAKARA company, 10 μ l linked systems, 16 DEG C of connections of spending the night.5 μ l connecting fluids are added in 50 μ lDH5a E. coli competent, 30min ice bath, 42 DEG C of thermal shock 90s, ice bath 3min, be applied to that to receive the solid agar sugar of resistance containing card dull and stereotyped, cultivate 14-16h for 37 DEG C.Picking mono-clonal is inoculated in be received in the LB liquid nutrient medium of resistance containing card, cultivates 12h, cellular lysate upgrading grain in the shaking table of 37 DEG C of 200rpm.Carrier sequence measures and is completed by Invitrogen company.
Table 3T4DNA linked system
Embodiment 2TALE-TFs shot design and TALE-TF plasmid
By sheep beta-casein gene sequence (X79703.1) and TALE(transcriptional activation sample effector) based on unit and DNA base-pair relationships, analysis and designation TALE-TFs(transcriptional activation sample effector-manual transcription factor) target sequence.TALE-TFs is synthesized by Guangzhou FulenGen Co., Ltd..Sheep beta-casein gene manual transcription factor target sequence comprises TALE-TF1, TALE-TF2, TALE-TF3, and concrete sequence is as follows:
TALE-TF1taatcatgcagatttctaaga
TALE-TF2tctaagaattcaaatccacta
TALE-TF3tccactattggttttatttca
ACAGAAGAATAGGAAGAATTCATTTCCTAATCATGCAGATTTCTAAGAATTCAAATCCACTATTGGTTTTATTTCAAACCACAAAATTAGCATGCCATTAAATAC
CAGCCACAAAATCAGATCATTATCCATTCAGCTTCTCCTTCACTTCTTCTCCTCTACTTTGGAAAAAAG
The RVD sequence that TALE-TF1 is corresponding is: NI-NI-NG-HD-NI-NG-NN-HD-NI-NN-NI-NG-NG-NG-HD-NG-NI-NI-NN-NI, TALE-TF1 plasmid dna sequence is SEQIDNO2.
The RVD sequence that TALE-TF2 is corresponding is: HD-NG-NI-NI-NH-NI-NI-NG-NG-HD-NI-NI-NI-NG-HD-HD-NI-HD-NG-NI, TALE-TF2 plasmid dna sequence is SEQIDNO3.
The RVD sequence that TALE-TF3 is corresponding is: HD-HD-NI-HD-NG-NI-NG-NG-NH-NH-NG-NG-NG-NG-NI-NG-NG-NG-HD-NI, TALE-TF3 plasmid dna sequence is SEQIDNO4.
The fibroblastic transfection of embodiment 3 sheep embryo
Step 1: sheep embryo inoblast obtains and Secondary Culture
Get the Chinese Merino super fine wool sheep tire sheep of pregnant 30 ages in days, adopt and organize fast culture method to cultivate, obtain sheep embryo inoblast.Cultivating the nutrient solution adopted is DMEM, adds 15% foetal calf serum, 100mg/L penicillin and 100mg/L Streptomycin sulphate, at 37 DEG C, 5%CO
2saturated humidity environment is cultivated.Changed nutrient solution 1 time every 2 days, within 4 days, go down to posterity 1 time.
When sheep embryo fibroblastic growth covers 80%-90% bottom culture dish, go down to posterity.Sheep embryo inoblast is washed 2 times, 0.25% trypsin solution digestion sheep embryo inoblast 1 ~ 3min by PBS solution.When cellular form becomes bowlder, add the nutrient solution containing serum 15%, stop cell dissociation.With pipettor pressure-vaccum gently, the sheep embryo inoblast to culture dish is completely floating.Sheep embryo inoblast suspension is transferred in centrifuge tube, the centrifugal 10min of 400 × g.Retain sheep embryo inoblast precipitation, supernatant discarded.Add the sheep embryo inoblast that 1ml nutrient solution fully suspends, proceed in Tissue Culture Dish by this suspension cell, add appropriate nutrient solution, rolling uniform cell, is placed in 37 DEG C, 5%CO
2cultivate in saturated humidity incubator.
Step 2: sheep embryo inoblast electrotransfection plasmid
According to 4D-Nucleofector electroporation (LonzaGroupLtd) operation instructions, 1:50 times of premix S1 solution (10ml solution: 2gATP-disodium salt, 1.2gMgCl
2* 6H
2and S2 solution (500ml solution: 6gKH O)
2pO
4, 0.6gNaHCO
3, 0.2g glucose) prepare electricity and turn liquid.Collect 2 × 10
6individual sheep embryo inoblast, liquid suspension cell is turned with 100ul electricity, add except endotoxic TALE-TF plasmid 2.5 μ g(TALE-TF1 plasmid, TALE-TF2 plasmid, TALE-TF3 plasmid) and except endotoxic red fluorescent protein reporter gene expression plasmid pShE1lB-Red-pA-ShE1RB or pShE2lB-Red-pA-ShE2RB2.5 μ g, proceed in electric shock cup.Electrotransfection positive control adopts pmaxFP-Green-N (LonzaGroupLtd, VDF-1012) plasmid, and the every 100 μ l electricity of 5 μ g turn liquid, detects transfection efficiency flow cytometer.Select CZ165 electricity carryover sequence, electricity is implemented to sheep embryo inoblast and turns.Leave standstill 10min, add the DMEM nutrient solution suspension cell containing 15% foetal calf serum, the sheep embryo inoblast after electrotransfection is proceeded to 6 well culture plates, 37 DEG C, 5.0%CO
2with saturated humidity CMC model, change nutrient solution after 6h 1 time.
Step 3:TALE-TFs activates Red reporter plasmid
Electricity turns cell 48h observation of cell fluorescence protein gene expression effect.With Laica inverted fluorescence microscope observation of cell.Observation of cell illumination effect under natural light, green fluorescence and red fluorescence light source, determines gene activation result respectively.
In order to determine that can TALE-TF1, TALE-TF2, TALE-TF3 tri-manual transcription factors activate beta-casein gene promotor, respectively by three manual transcription factors and Red reporter plasmid pShE1lB-Red-pA-ShE1RB or pShE2lB-Red-pA-ShE2RB cotransfection Sheep Fibroblast.The egfp expression (as shown in Figure 6) can observed during electrotransfection 36h, illustrates three manual transcription factors all normal expressions.During 72h and 96h, in the Sheep Fibroblast of TALE-TF1, TALE-TF2 and TALE-TF3 tri-manual transcription factor transfections, red fluorescent protein can be observed and express (as shown in Figure 7).Illustrate in Sheep Fibroblast, these three manual transcription factors can activate the goal gene plasmid of beta-casein gene promoter regulation.
Step 4:TALE-TFs activates Sheep Fibroblast beta-casein gene result
Liquid [S1 solution (10ml solution: 2gATP-disodium salt, 1.2gMgCl is turned with the TALE-TFs100 μ lNucleoeffector electricity of interpolation 5 μ g
2* 6H
2and S2 solution (500ml solution: 6gKH O)
2pO
4, 0.6gNaHCO
3, 0.2g glucose), prepare electricity by S1:S2=1:50 volume ratio and turn liquid], suspend 2 × 10
6individual sheep embryo inoblast, be inoculated in 6 porocyte culture plates after electrotransfection, 12h changes liquid 1 time, cultivates 72h, with 1mlTrizol(Invitrogen, 10296-010) By Direct Pyrolysis cell, extract total serum IgE.
The PrimeScriptTMRTreagentKitwithgDNAEraser(RR047A of application TAKARA company) process total serum IgE, 42 DEG C of incubation 2min, the genomic dna that removing is residual.Eliminate genomic dna reaction system residual in RNA for listed by table 4.
Genomic dna reaction system residual in RNA eliminated by table 4
Adopt
iIIFirst-StrandSynthesisSystemforRT-PCR test kit (Invitrogen company, 18080-051) prepares cDNA.
Mixed by following solution, 65 DEG C of 5min, cooled on ice 2min, realize RNA sex change.
Table 5RNA reaction of degeneration system and reverse transcription system
Add the cDNASynthesisMix that 7 μ l tables 6 are listed, mixing, 37 DEG C of standing 2min.
Table 6cDNASynthesisMix reagent
Room temperature adds 1 μ lMultiScribe
tMmuLV reversed transcriptive enzyme.25℃10min，37℃50min。The cDNA solution-20 DEG C obtained is frozen, as the DNA masterplate of fluorescence real-time quantitative PCR.
Application quantitative PCR determines that TALE-TFs activates the beta-casein gene transcriptional efficiency in sheep embryo inoblast.Adopt Roche LightCycler480 real-time PCR, TAKARA company
premixExTaq
tM(TliRNaseHPlus) (RR420A) fluorescence quantitative kit, primer adopts ShGAPDHRTF/ShGAPDHRTR and ShbetaRTF/ShbetaRTR.Reaction system is as follows.
Table 7 quantitative fluorescent PCR reaction system
Sex change: 95 DEG C of 30s(temperature rise rates, 4.4 DEG C/s), 1cycle;
PCR quantitative analysis pattern: 95 DEG C of 5s (temperature rise rate 4.4 DEG C/s), 60 DEG C of 30s (temperature rise rate 2.2 DEG C/s, AcquisitionMode:Single), 45cycles;
Melt curve analysis analytical model: 95 DEG C of 5s (temperature rate 4.4 DEG C/s), 60 DEG C of 1min (temperature rate 2.2 DEG C/s), 95 DEG C of (temperature rate 0.11 DEG C/s, AcquisitionMode:Continuous, Acquisitions:5per DEG C), 1cycle;
Cooling: 50 DEG C of 30s (temperature rate 2.2 DEG C/s), 1cycle.
Reverse transcription RT-PCR result (shown in Fig. 8) and fluorescence real-time quantitative PCR result (shown in Fig. 9) illustrate that TALE-TF1, TALE-TF2 and TALE-TF3 tri-manual transcription factors effectively can activate the beta-casein gene in Sheep Fibroblast genome.
Above embodiment is only exemplary embodiment of the present invention, and be not used in restriction the present invention, protection scope of the present invention is defined by the claims.Those skilled in the art can in essence of the present invention and protection domain, and make various amendment or equivalent replacement to the present invention, this amendment or equivalent replacement also should be considered as dropping in protection scope of the present invention.
Claims (5)
1. can encode the artificial DNA molecule of TALE-TFs, and it is characterized in that, the base sequence of described artificial DNA molecule is SEQIDNO.2, and can encode TALE-TF1, and described TALE-TF1 can identify the 28-48 position in SEQIDNO.1; Or the base sequence of described artificial DNA molecule is SEQIDNO.3, can encode TALE-TF2, and described TALE-TF2 can identify the 42-62 position in SEQIDNO.1; Or the base sequence of described artificial DNA molecule is SEQIDNO.4, can encode TALE-TF3, and described TALE-TF3 can identify the 56-76 position in SEQIDNO.1.
2. an expression vector, is characterized in that, described expression vector comprises artificial DNA molecule according to claim 1, and it can express TALE-TF1, TALE-TF2 or TALE-TF3.
3. a reconstitution cell, is characterized in that, described reconstitution cell uses the prokaryotic cell prokaryocyte or eukaryotic cell that described in claim 2, expression vector transforms.
4. express a method for target gene, it is characterized in that,
Steps A, target gene is proceeded to sheep beta-casein gene, build the sheep beta-casein gene expression vector containing target gene;
Step B, according to sheep beta-casein gene promotor target sequence design TALE arrangement, and the expression vector of construction expression TALE-TFs; Wherein said sheep beta-casein gene promotor target sequence is the 28-48 position in SEQIDNO.1, or target sequence is the 42-62 position in SEQIDNO.1, or target sequence is the 56-76 position in SEQIDNO.1;
Step C, obtain the Sheep Fibroblast after transfection with the expression vector cotransfection Sheep Fibroblast of described sheep beta-casein gene expression vector containing target gene and described expression TALE-TFs.
5. the method for expression target gene according to claim 4, is characterized in that, the expression vector of expressing TALE-TFs described in step B comprises SEQIDNO.2 base sequence, or SEQIDNO.3 base sequence, or SEQIDNO.4 base sequence.
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| CN102558309A (en) * | 2012-02-10 | 2012-07-11 | 浙江大学 | Transcription activator-like effector nucleases, and encoding genes and application thereof |
| CN103131695A (en) * | 2013-02-07 | 2013-06-05 | 西南大学 | Efficient assembling method of transcription activator-like effectors (TALE) repeating region for editing silkworm genome and framework carrier thereof |
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| CN102558309A (en) * | 2012-02-10 | 2012-07-11 | 浙江大学 | Transcription activator-like effector nucleases, and encoding genes and application thereof |
| CN103131695A (en) * | 2013-02-07 | 2013-06-05 | 西南大学 | Efficient assembling method of transcription activator-like effectors (TALE) repeating region for editing silkworm genome and framework carrier thereof |
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| Title |
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| A transcription activator-like effector toolbox for genome engineering;Neville E Sanjana等;《Nature Protocol》;20120105;第7卷(第1期);第172页图1，第172页左栏倒数第1段至第173页左栏倒数第1段，第173页右栏倒数第1段至第174页左栏第1段，第173页图1b，第181页第5段，第186页图6 * |
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