Platinum gate talen kit

Both Platinum TALENs and CRISPR/Cas9 (i.e., gRNA) were designed to target the sequence of exon 3 in the pig SALL1 gene (Fig. 1A). The SALL1-targeted Platinum TALEN plasmids were constructed using the Platinum Gate TALEN Kit (Addgene Kit #1000000043) developed by Sakuma et al.^{53 (link)}. The Platinum TALEN plasmids to be used as templates for in vitro transcription (IVT) were linearized, and then capped and poly (A)-tailed Platinum TALEN mRNAs were synthesized by using an mMESSAGE mMACHINE T7 Ultra kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. For the CRISPR/Cas9 system, four nucleotide spacers (GCGT) and the T7 promoter were added to the gRNA template by polymerase chain reaction (PCR) amplification using the following primers: 5′-GCGTTAATACGACTCACTATAGGGCCGCCCAGGCCGGCAACC and 5′-AAAAGCACCGACTCGGTGCC. The amplified template was gel-purified and used as the template for IVT using a MEGAshortscript T7 kit (Thermo Fisher Scientific). Cas9 mRNA was synthesized by the methods described above using pCAG-hCas9 (Addgene #51142) as a template for IVT. Cas9 protein was obtained from Takara Bio (Shiga, Japan). The Platinum TALEN-encoding mRNAs, gRNA and Cas9 mRNA were purified using a MEGAclear kit and eluted into RNase-free water. These genome-editing molecules were stored at −80 °C until cytoplasmic injection.

A two-step Golden Gate assembly method using the Platinum Gate TALEN Kit (Addgene; cat#1000000043) [15 (link)] was used to construct Platinum TALEN plasmids containing the homodimer-type FokI nuclease domain. The assembled repeat arrays were subsequently inserted into the final destination vector, ptCMV-153/47-VR. For double knock-out experiments, previously reported TALENs for pax6 were used [12 (link)]. Target sequences for TALENs are summarized in S1 Table.

TALEN plasmids were assembled using the Platinum Gate TALEN kit (Addgene; catalog number 1000000043) as described previously (32 ). The ptCMV-153/47-VR-HD and ptCMV-153/47-VR-NG vectors were used as destination vectors for the left and right TALENs, respectively. Activity of constructed TALEN plasmids were validated by the human cell-based single-strand annealing assay as previously described (33 (link)). TALEN mRNAs were synthesized from plasmid DNA encoding TRα TALENs (right and left arms) linearized with XmaI followed by lithium chloride precipitation using T7 mMESSAGE mMACHINE (Life Technologies). To monitor embryo injections, mCherry mRNA from KpnI-linearized CS108-mCherry (a gift from Dr M. Khokha) was also prepared with T7 and lithium chloride precipitation. A mixture of TRα TALEN mRNAs (400 pg for the left arm, 400 pg or the right arm), and mCherry mRNAs (25 pg) were injected into each embryo.

Gene disruption was performed with the TALEN system. TALEN plasmids were constructed using the Platinum Gate TALEN Kit (Addgene, Cambridge, MA, USA; Kit #1000000043) as described previously[17 (link)] with modifications described as follows: the plasmid backbone of ptCMV-136/63-VR vectors were replaced with pCS2P, containing SP6 promoter for in vitro transcription. Medaka ptena and ptenb TALEN constructs were digested with NotI (Thermo Fisher Scientific, Waltham, MA, USA) and were transcribed in vitro with the use of a mMessage mMachine SP6 transcription kit (Thermo Fisher Scientific).

Plasmid DNA, CSII-EF-EGFP, was constructed from CSII-EF-RfA, which was a kind gift from Dr. Hiroyuki Miyoshi (RIKEN BioResource Center, Tsukuba, Japan). TALEN plasmid DNA driven by the human elongation factor 1-alpha 1 (EF1α) promoter was constructed as described previously^{24 (link)} with some modifications. Briefly, DNA-binding modules were assembled with the two-step Golden Gate assembly method using the Platinum Gate TALEN Kit (Addgene). The CMV promoter of ptCMV-136/63-VR vector was replaced with the EF1α promoter. Dissociated hPSCs were seeded at a density of 1.2 × 10⁵/well in MATRIX-coated 12-well plates and cultured for 24 h. Both 1 μg of plasmid DNA and 4 μL of FuGENE HD (Promega) were diluted in 100 μL of Opti-MEM I Reduced Serum Media (Life Technologies) and incubated for 15 min at room temperature. This mixture was transferred to one well of a 12-well plate. Twenty-four hours post-transfection, cells were analyzed for enhanced green fluorescent protein (eGFP) expression to determine transfection efficiency. GeneJammer (Agilent Technologies), X-tremeGENE HP (Roche), and Lipofectamine 2000 (Invitrogen) were used following the manufacturers' instructions. Transfected cells were observed under a fluorescence microscope (BZ-9000; Keyence) and analyzed using BZ-Analyzer software (Keyence).

We used E. coli strain NEB stable (New England Biolab, MA, USA) that is expected to reduce unexpected recombination in repeat sequences for the plasmid construction. Platinum Gate TALEN kit was obtained from Addgene (#1000000043, Cambridge, MA, USA).

FLAG-tagged TALEN plasmids were constructed with the two-step Golden Gate cloning method using a Platinum Gate TALEN Kit (Addgene; cat#1000000043) as previously described [21 (link)]. A ptCMV-153/47-VR-NI vector (Addgene; Plasmid 50705) was used for the destination vector. Left and right TALEN target sequences and assembled RVD modules are shown in S1 Table.