Anti-FLAG antibody was purchased from Sigma (St. Louis, MO); anti-HA and anti-phospho-IκBα antibodies were purchased from Cell Signaling (Danvers, MA); anti-β-actin antibody was purchased from Abcam (Cambridge, MA); anti-A20 antibody was purchased from Ebioscience (San Diego, CA). HEK293T cell line was purchased and maintained per instructions from American Type Culture Collection (ATCC; Manassas, VA). Golden Gate cloning kit and expression vectors, pCS2TAL3DD and pCS2TAL3RR, were purchased from Addgene (Cambridge, MA). DH5α competent cells were purchased from Life Technologies (Grand Island, NY). QuantiTect SYBR Green PCR Master Mix was purchased from QIAGEN (Valencia, CA). Restriction enzymes NlaIII, EcoRV, and NEB Standard Buffer 4 were purchased from New England Biolabs (Ipswich, MA).
Pcs2tal3dd
Manufactured by Addgene
The PCS2TAL3DD is a lab equipment product. It is designed for core functions related to laboratory operations. However, a detailed description of its functionality cannot be provided while maintaining an unbiased and factual approach. Additional information about its intended use or capabilities is not available.
Automatically generated - may contain errors
Lab products found in correlation
Pcs2tal3rr, by Addgene (9 mentions)
Dh5α competent cells, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Hek293t cell line, by American Type Culture Collection (2 mentions)
Anti flag antibody, by Merck Group (2 mentions)
Quantitect sybr green pcr master mix, by Qiagen (2 mentions)
Anti phospho iκbα, by Cell Signaling Technology (2 mentions)
Ecorv, by New England Biolabs (2 mentions)
Anti ha, by Cell Signaling Technology (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Anti a20 antibody, by Thermo Fisher Scientific (2 mentions)
Neb standard buffer 4, by New England Biolabs (2 mentions)
Nlaiii, by New England Biolabs (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Tal effector kit, by Addgene (2 mentions)
Talen golden gate assembly system, by Addgene (2 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (2 mentions)
Dh5a competent cells, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using pcs2tal3dd
1
A20 Regulation of NF-κB Signaling
2
TALEN Design and Construction for pcgf1 Gene
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The pcgf1 TALEN target site was selected using the online TAL Effector-Nucleotide Targeter tool (https://tale-nt.cac.cornell.edu/ ; [35 (link)]) in exon 2 with the following parameters: (i) spacer length of 14–17 bp, (ii) repeat array length of 16–18 bp, (iii) each binding site was anchored by a preceding T base in position ‘‘0” as has been shown to be optimal for naturally occurring TAL proteins [36 (link), 37 (link)], (iv) presence of a restriction site (ClaI) within the spacer sequence for screening and genotyping purposes.
Pcgf1-specific TALEN constructs were engineered using the TALEN Golden Gate assembly system described by Cermak et al., [38 (link)]. The TALEN expression backbones, pCS2TAL3DD and pCS2TAL3RR [27 (link)], and the plasmids providing repeat variable diresidues (RVD) [38 (link)] for Golden Gate Cloning were obtained from Addgene.
Pcgf1-specific TALEN constructs were engineered using the TALEN Golden Gate assembly system described by Cermak et al., [38 (link)]. The TALEN expression backbones, pCS2TAL3DD and pCS2TAL3RR [27 (link)], and the plasmids providing repeat variable diresidues (RVD) [38 (link)] for Golden Gate Cloning were obtained from Addgene.
3
TALEN Design for Targeting kif6 Exon
4
TALE-SET Fusion Protein Construction
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The FokI domain of the TALE-FokI expression vector (pCS2TAL3-DD, Addgene) was replaced with the SET catalytic domain of EHMT2 using the restriction enzymes BamHI and XbaI such that SET was fused to the C-terminal end of the TAL effector. TALE repeat arrays were designed and assembled via multi-fragment cut-ligation using Golden Gate cloning [27 (link)]. These assembled DNA fragments were cloned into the TSET expression vector and the construct was verified by restriction enzyme digestion and sequencing. A 40-amino acid ((GGGS)10) linker was inserted between the TAL effector repeats and the SET domain.
5
Targeted Genomic Editing with TALENs
6
A20 Regulation of NF-κB Signaling
7
TALEN-Mediated Editing of ctnna1 Locus
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TALENs targeting the ctnna1 locus were designed using the TALE-NT tool (https://tale-nt.cac.cornell.edu/node/add/talen ) using the guidelines specified in Cermak et al. (2011) (link). The chosen optimal target sequence in exon 3: 5′- TTGTGTTTATTGCAGG TCACCACACTTGTGAACTCCAGCAACAAAGGTCCA -3′ (binding sites of the left and right TALEN arms are underlined) contains a HphI (NEB) restriction enzyme site (GGTGA). TALENs were generated using the Golden Gate kit (Cermak et al., 2011 (link)) in combination with the obligate heterodimeric FokI pCS2TAL3DD (Addgene, #37275) and pCS2TAL3RR (Addgene, #37276) backbones (Ota et al., 2013 (link)).
TALEN targeting plasmids were linearized with NotI (Promega) after which IVT mRNA was generated using the mMessage mMachine SP6 kit (Ambion), and purified using the RNeasy mini kit (Qiagen).
TALEN targeting plasmids were linearized with NotI (Promega) after which IVT mRNA was generated using the mMessage mMachine SP6 kit (Ambion), and purified using the RNeasy mini kit (Qiagen).
8
TALEN Design and Synthesis for vcla
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TALENs targeting the vcla locus were designed using the TALE-NT tool (https://tale-nt.cac.cornell.edu/node/add/talen ) using the guidelines specified in [41 (link)]. The chosen optimal target sequence in exon 4:
5’-TTGTGGAAACCATGGAGG ACTTGATCACTTACACTAAAAACCTGGGACCAGGTA -3’ (binding sites of the Left and Right TALEN arms are underlined) contains a BclI (Promega) restriction enzyme site (TGATCA). TALENs were generated using the Golden Gate kit in combination with the obligate heterodimeric FokI pCS2TAL3DD (Addgene, #37275) and pCS2TAL3RR (Addgene, #37276) backbones [42 (link)].
TALEN targeting plasmids were linearized with NotI (Promega) after which IVT mRNA was generated using the mMessage mMachine SP6 kit (Ambion), and purified using the RNeasy mini kit (Qiagen).
TALEN targeting plasmids were linearized with NotI (Promega) after which IVT mRNA was generated using the mMessage mMachine SP6 kit (Ambion), and purified using the RNeasy mini kit (Qiagen).
9
Targeted Genomic Editing with TALENs
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Genomic DNA sequence surrounding the TT>A enhancer was scanned for potential TALEN target sites using TALEN-Targeter 2.0 (27 (link)). A modified TALEN Golden Gate assembly system (Addgene, Cambridge, MA) was then used to generate repeat variable di-residue (RVD) arrays to target the TT>A enhancer. The RVD sequence was subcloned into the expression vectors pCS2TAL3DD (5’ arm) and pCS2TAL3RR (3’ arm) (Addgene, Cambridge MA). Sanger sequencing confirmed the correct insertion into each expression vector (31 (link), 32 (link)). TALENs were expressed in HEK293T cells by transient transfection using Fugene HD (VWR, Radnor, PA) with 2 µg of each plasmid DNA encoding HA-tagged 5’-arm and FLAG-tagged 3’-arm. After 48 h, protein expressions of 5’ and 3’ TALEN arms were detected using Western blotting with antibodies against the respective HA (5’ arm) and FLAG (3’ arm) tags. Single cell clones of the HEK293T cells expressing TALENs were isolated and genomic DNA harvested by phenol-chloroform extraction. The genomic DNA of the TT>A enhancer in each cell clone was amplified with PCR (Supplemental Table 1 ) and screened using high-resolution melting curve analysis (28 (link)). PCR amplicon sequences from each clone were further validated by Sanger sequencing (31 (link), 32 (link)).
10
TALEN-mediated Genome Editing in Zebrafish
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Left and right TALENs were designed to target the sites flanking codon 673 of smyhc1, on exon 16. TALENs were generated using the Golden Gate method as previously described (Cermak et al, 2011 ). Plasmids were acquired from the Golden Gate TALEN and TAL Effector Kit (AddGene) and RVD repeat arrays were cloned into pCS2TAL3DD and pCS2TAL3RR (AddGene). Plasmids were transformed into DH5α competent cells (Invitrogen) and isolated using the QIAprep Spin Miniprep Kit (Qiagen).
Subsequently, plasmids encoding the scaffold and RVD arrays were linearized via restriction digest and 5′‐capped mRNA was generated by in vitro transcription using the mMESSAGE mMACHINE SP6 Transcription Kit (Life Technologies). Capped mRNA was purified using the RNeasy Mini Kit (Qiagen). WT embryos were collected, and 40–50 pg of pooled left and right TALENs at equal concentrations were injected into the yolk of 1–4 cell stage embryos. Embryos were raised to adulthood, and sperm and progeny of these fish were collected and sequenced with MiSeq to screen for germline transmission.
Subsequently, plasmids encoding the scaffold and RVD arrays were linearized via restriction digest and 5′‐capped mRNA was generated by in vitro transcription using the mMESSAGE mMACHINE SP6 Transcription Kit (Life Technologies). Capped mRNA was purified using the RNeasy Mini Kit (Qiagen). WT embryos were collected, and 40–50 pg of pooled left and right TALENs at equal concentrations were injected into the yolk of 1–4 cell stage embryos. Embryos were raised to adulthood, and sperm and progeny of these fish were collected and sequenced with MiSeq to screen for germline transmission.
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