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ptale tf v2 ni (Addgene inc)

Name: ptale tf v2 ni
Brand: Addgene inc
Rating: 93 (2 reviews)

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Addgene inc ptale tf v2 ni
Ptale Tf V2 Ni, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 2 article reviews

ptale tf v2 ni - by Bioz Stars, 2026-02

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(a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA binding module consists of 34 amino acids, where the repeat variable diresidues (RVDs) in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG = T, HD = C, NI = A, and NN = G or A. The DNA binding modules are flanked by non-repetitive amino and carboxyl termini, which carry the translocation, nuclear localization (NLS), and transcription activation (AD) domains. A cryptic signal within the amino terminus specifies a thymine as the first base of the target site. (b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids in total: 4 monomer plasmids to be used as templates for PCR amplification, 4 TALE-TF and 4 TALEN cloning backbones corresponding to 4 different bases targeted by the 0.5 repeat. CMV: cytomegalovirus promoter; N-term: non-repetitive amino terminus from the Hax3 TALE; C-term: non-repetitive carboxyl terminus from the Hax3 TALE; BsaI: type IIs restriction sites used for the insertion of custom TALE DNA binding domains; ccdB+CmR: negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene; NLS: nuclear localization signal; VP64: synthetic transcriptional activator derived from VP16 protein of herpes simplex virus; 2A: 2A self-cleavage linker; EGFP: enhanced green fluorescent protein; polyA signal: polyadenylation signal; FokI: catalytic domain from the FokI endonuclease. (c) TALEs can be used to generate custom transcription factors (TALE-TFs) and modulate the transcription of endogenous genes from the genome. This schematic shows a TALE-TF designed to target the SOX2 locus in the human genome. The SOX2 TALE-TF recognizes the sense strand of the SOX2 proximal promoter, and the recognition site begins with T. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription. (d) TALE nucleases (TALENs) can be used to generate site-specific double strand breaks to facilitate genome editing through non-homologous repair or homology-directed repair. This schematic shows a pair of TALENs designed to target the AAVS1 locus in the human genome. Two TALENs target a pair of binding sites flanking a 16bp spacer. The left and right TALENs recognize the top and bottom strands of the target sites respectively. Each TALE DNA-binding domain is fused to the catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right TALEN binding sites.

Journal: Nature protocols

Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

doi: 10.1038/nprot.2011.431

Figure Lengend Snippet: (a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA binding module consists of 34 amino acids, where the repeat variable diresidues (RVDs) in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG = T, HD = C, NI = A, and NN = G or A. The DNA binding modules are flanked by non-repetitive amino and carboxyl termini, which carry the translocation, nuclear localization (NLS), and transcription activation (AD) domains. A cryptic signal within the amino terminus specifies a thymine as the first base of the target site. (b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids in total: 4 monomer plasmids to be used as templates for PCR amplification, 4 TALE-TF and 4 TALEN cloning backbones corresponding to 4 different bases targeted by the 0.5 repeat. CMV: cytomegalovirus promoter; N-term: non-repetitive amino terminus from the Hax3 TALE; C-term: non-repetitive carboxyl terminus from the Hax3 TALE; BsaI: type IIs restriction sites used for the insertion of custom TALE DNA binding domains; ccdB+CmR: negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene; NLS: nuclear localization signal; VP64: synthetic transcriptional activator derived from VP16 protein of herpes simplex virus; 2A: 2A self-cleavage linker; EGFP: enhanced green fluorescent protein; polyA signal: polyadenylation signal; FokI: catalytic domain from the FokI endonuclease. (c) TALEs can be used to generate custom transcription factors (TALE-TFs) and modulate the transcription of endogenous genes from the genome. This schematic shows a TALE-TF designed to target the SOX2 locus in the human genome. The SOX2 TALE-TF recognizes the sense strand of the SOX2 proximal promoter, and the recognition site begins with T. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription. (d) TALE nucleases (TALENs) can be used to generate site-specific double strand breaks to facilitate genome editing through non-homologous repair or homology-directed repair. This schematic shows a pair of TALENs designed to target the AAVS1 locus in the human genome. Two TALENs target a pair of binding sites flanking a 16bp spacer. The left and right TALENs recognize the top and bottom strands of the target sites respectively. Each TALE DNA-binding domain is fused to the catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right TALEN binding sites.

Article Snippet: REAGENTS TALE construction list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 TALE monomer template plasmids: list-behavior=simple prefix-word= mark-type=none max-label-size=0 pNI_v2 pNG_v2 pNN_v2 pHD_v2 TALE transcriptional activator (TALE-TF) plasmids: list-behavior=simple prefix-word= mark-type=none max-label-size=0 pTALE-TF_v2 (NI) pTALE-TF_v2 (NG) pTALE-TF_v2 (NN) pTALE-TF_v2 (HD) TALE nuclease (TALEN) backbone plasmids: list-behavior=simple prefix-word= mark-type=none max-label-size=0 pTALEN_v2 (NI) pTALEN_v2 (NG) pTALEN_v2 (NN) pTALEN_v2 (HD) These plasmids can be obtained individually or bundled together as a single kit from the Zhang Lab plasmid collection at Addgene ( http://www.addgene.org/TALE_Toolbox ).

Techniques: Derivative Assay, Binding Assay, Translocation Assay, Activation Assay, TALENs, Amplification, Cloning, Selection, Virus

Applications of custom TALEs on endogenous genome targets

Journal: Nature protocols

Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

doi: 10.1038/nprot.2011.431

Figure Lengend Snippet: Applications of custom TALEs on endogenous genome targets

Techniques:

Schematic of the construction process for a custom TALE containing a 18-mer tandem repeat DNA binding domain. Stage 1: specific primers are used to amplify each monomer and add the appropriate ligation adapters (Procedure Steps 1–9). Stage 2: hexameric tandem repeats (1—6, 7—12, and 13—18) are assembled first using Golden Gate digestion-ligation. The 5′ ends of monomers 1, 7, and 13 and the 3′ ends of monomers 6, 12, and 18 are designed so that each tandem hexamer assembles into an intact circle (Procedure Steps 10–15). Stage 3: the Golden Gate reaction is treated with an exonuclease to remove all linear DNA, leaving only the properly assembled tandem hexamer (Procedure Steps 16–17). Stage 4: each tandem hexamer is amplified individually using PCR and purified (Procedure Steps 18–25). Stage 5: tandem hexamers corresponding to 1—6, 7—12, and 13—18 are ligated into the appropriate TALE-TF or TALEN cloning backbone using Golden Gate cut-ligation (Procedure Steps 26–28). Stage 6: The assembled TALE-TF or TALEN is transformed into competent cells and successful clones are isolated and sequence verified (Procedure Steps 29–38).

Journal: Nature protocols

Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

doi: 10.1038/nprot.2011.431

Figure Lengend Snippet: Schematic of the construction process for a custom TALE containing a 18-mer tandem repeat DNA binding domain. Stage 1: specific primers are used to amplify each monomer and add the appropriate ligation adapters (Procedure Steps 1–9). Stage 2: hexameric tandem repeats (1—6, 7—12, and 13—18) are assembled first using Golden Gate digestion-ligation. The 5′ ends of monomers 1, 7, and 13 and the 3′ ends of monomers 6, 12, and 18 are designed so that each tandem hexamer assembles into an intact circle (Procedure Steps 10–15). Stage 3: the Golden Gate reaction is treated with an exonuclease to remove all linear DNA, leaving only the properly assembled tandem hexamer (Procedure Steps 16–17). Stage 4: each tandem hexamer is amplified individually using PCR and purified (Procedure Steps 18–25). Stage 5: tandem hexamers corresponding to 1—6, 7—12, and 13—18 are ligated into the appropriate TALE-TF or TALEN cloning backbone using Golden Gate cut-ligation (Procedure Steps 26–28). Stage 6: The assembled TALE-TF or TALEN is transformed into competent cells and successful clones are isolated and sequence verified (Procedure Steps 29–38).

Techniques: Binding Assay, Ligation, Amplification, Purification, Cloning, Transformation Assay, Clone Assay, Isolation, Sequencing

(a) Lanes 1—6: products from the monomer PCR reaction (Stage 1 in Figure 3) after purification and gel normalization (Procedure Steps 8–9). The molar concentrations of samples shown on this gel have been normalized so that equal moles of monomers are mixed for downstream steps. Monomers 1 and 6 are slightly longer than monomers 2, 3, 4, and 5 due to the addition of sequences used for circularization. Lane 7: result of the hexamer Golden Gate cut-ligation (Procedure Step 15). A series of bands with size ~700 bp and lower can be seen. Successful hexamer Golden Gate assembly should show a band ~700 bp (as indicated by arrow). Lane 8: hexamer assembly after PlasmidSafe exonuclease treatment (Procedure Step 17). Typically the amount of circular DNA remaining is difficult to visualize by gel. Lane 9: result of hexamer amplification (Procedure Step 20). A ~700 bp band should be clearly visible. The hexamer gel band should be gel-purified to remove shorter DNA fragments. (b) Properly assembled TALE-TFs and TALENs can be verified using bacterial colony PCR (2175 bp band, lane 1) (Procedure Step 35) and restriction digest with AfeI (2118 bp band for correctly assembled 18-mer in either backbone; other bands for TALE-TF are 165 bp, 3435 bp, 3544 bp; other bands for TALEN are 165 bp, 2803 bp, 3236 bp; digest shown is for TALE-TF backbone vector, lane 2) (Procedure Step 35).

Journal: Nature protocols

Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

doi: 10.1038/nprot.2011.431

Figure Lengend Snippet: (a) Lanes 1—6: products from the monomer PCR reaction (Stage 1 in Figure 3) after purification and gel normalization (Procedure Steps 8–9). The molar concentrations of samples shown on this gel have been normalized so that equal moles of monomers are mixed for downstream steps. Monomers 1 and 6 are slightly longer than monomers 2, 3, 4, and 5 due to the addition of sequences used for circularization. Lane 7: result of the hexamer Golden Gate cut-ligation (Procedure Step 15). A series of bands with size ~700 bp and lower can be seen. Successful hexamer Golden Gate assembly should show a band ~700 bp (as indicated by arrow). Lane 8: hexamer assembly after PlasmidSafe exonuclease treatment (Procedure Step 17). Typically the amount of circular DNA remaining is difficult to visualize by gel. Lane 9: result of hexamer amplification (Procedure Step 20). A ~700 bp band should be clearly visible. The hexamer gel band should be gel-purified to remove shorter DNA fragments. (b) Properly assembled TALE-TFs and TALENs can be verified using bacterial colony PCR (2175 bp band, lane 1) (Procedure Step 35) and restriction digest with AfeI (2118 bp band for correctly assembled 18-mer in either backbone; other bands for TALE-TF are 165 bp, 3435 bp, 3544 bp; other bands for TALEN are 165 bp, 2803 bp, 3236 bp; digest shown is for TALE-TF backbone vector, lane 2) (Procedure Step 35).

Techniques: Purification, Ligation, Amplification, TALENs, Plasmid Preparation

$(a) Schematic of the Surveyor nuclease assay used to determine TALEN cleavage efficiency. First, genomic PCR is used to amplify the TALEN target region from a heterogeneous population of TALEN-modified and unmodified cells, and the gPCR products are re-annealed slowly to generate heteroduplexes. The re-annealed heteroduplexes are cleaved by Surveyor nuclease while homoduplexes are left intact. TALEN cleavage efficiency is calculated based on the fraction of cleaved DNA. (b) Gel showing the Surveyor nuclease result from the AAVS1 TALEN pair (from Fig. 1d). Lanes 1—4: controls from un-transfected (N.T.) cells and cells transfected with a plasmid carrying GFP (Mock), AAVS1 left TALEN only (L), and AAVS1 right TALEN only (R). Lanes 5—7: cells transfected with AAVS1 Left and Right TALENs (L+R) for 24, 48, and 72 hours. The two lower bands indicated by the arrows are Surveyor-cleaved DNA products. (c) 293FT cells transfected with the SOX2 TALE-TF (from Fig. 1c) exhibited a 5 fold increase in the amount of SOX2 mRNA compared with mock transfected cells. Error bars indicate s.e.m.; n = 3. *** indicates P < 0.005. Panel c was modified with permission from Nature Biotechnology3 (Nature Biotechnology (c) 2011, Macmillian Publishers Ltd.).$

Journal: Nature protocols

Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

doi: 10.1038/nprot.2011.431

Figure Lengend Snippet: (a) Schematic of the Surveyor nuclease assay used to determine TALEN cleavage efficiency. First, genomic PCR is used to amplify the TALEN target region from a heterogeneous population of TALEN-modified and unmodified cells, and the gPCR products are re-annealed slowly to generate heteroduplexes. The re-annealed heteroduplexes are cleaved by Surveyor nuclease while homoduplexes are left intact. TALEN cleavage efficiency is calculated based on the fraction of cleaved DNA. (b) Gel showing the Surveyor nuclease result from the AAVS1 TALEN pair (from Fig. 1d). Lanes 1—4: controls from un-transfected (N.T.) cells and cells transfected with a plasmid carrying GFP (Mock), AAVS1 left TALEN only (L), and AAVS1 right TALEN only (R). Lanes 5—7: cells transfected with AAVS1 Left and Right TALENs (L+R) for 24, 48, and 72 hours. The two lower bands indicated by the arrows are Surveyor-cleaved DNA products. (c) 293FT cells transfected with the SOX2 TALE-TF (from Fig. 1c) exhibited a 5 fold increase in the amount of SOX2 mRNA compared with mock transfected cells. Error bars indicate s.e.m.; n = 3. *** indicates P < 0.005. Panel c was modified with permission from Nature Biotechnology3 (Nature Biotechnology (c) 2011, Macmillian Publishers Ltd.).

Techniques: Nuclease Assay, Modification, Transfection, Plasmid Preparation, TALENs

Journal: Nature protocols

Article Title: A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

doi: 10.1038/nprot.2011.431

Figure Lengend Snippet: Troubleshooting.

Techniques: Amplification, Concentration Assay, Purification, Binding Assay, Gel Extraction, Transformation Assay, Negative Control, Suspension, Clone Assay, Transfection, Plasmid Preparation, Touchdown PCR

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