To generate the miC vectors, we searched for sequences in intron 1, exon 2, and exon 11 of C9orf72 that were mostly conserved among human and non-human primates and mouse. The miC sequences were incorporated into the cellular pri-miRNA miR-101-1 or miR-451 scaffold of the human. 200-nt 5′- and 3′-flanking regions were included with EcoRV and BamHI restriction sites, and the mfold program (http://unafold.rna.albany.edu/?q=mfold ) was used to determine if the miC candidates were folded correctly into their secondary structures. The complete sequences were ordered from GeneArt gene synthesis (Invitrogen). These constructs were subsequently cloned into an expression vector containing the CMV immediate-early enhancer fused to chicken β-actin (CAG) promoter (Inovio, Plymouth Meeting, PA) using the EcoRV and BamHI sites. For generation of the Luc reporters, sequences from C9orf72 intron 1 (sense and antisense), exon 2, or exon 11 were synthesized at GeneArt gene synthesis and cloned in the 3′ UTR of the RL gene of the psiCHECK-2 vector (Promega, Madison, WI). The FL gene was also expressed in this vector and served as an internal control (Figure 2 D).
Geneart gene synthesis
Manufactured by Promega
Sourced in United States
GeneArt gene synthesis is a laboratory tool that allows for the custom design and synthesis of DNA sequences. It provides the ability to create specific genetic constructs for various research and application purposes.
Automatically generated - may contain errors
Lab products found in correlation
Geneart gene synthesis, by Thermo Fisher Scientific (3 mentions)
Psicheck 2 vector, by Promega (2 mentions)
Pnl1.2 nlucp, by Promega (1 mentions)
Kld enzyme mix, by New England Biolabs (1 mentions)
Nanodrop nd 1000, by Macherey-Nagel (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Nucleospin plasmid transfection grade, by Macherey-Nagel (1 mentions)
3 protocols using geneart gene synthesis
1
Generating miC Vectors for C9orf72 Gene Study
2
Generating miATXN3 Vectors and Reporters
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To generate the miATXN3 vectors, we searched for sequences on the ATXN3 gene that were mostly conserved between humans, non-human primates, and rodents. The sequences were incorporated into the cellular miR-451 scaffold of humans. 200-nt 5′ and 3′ flanking regions were included with EcoRV and BamHI restriction sites, and the mfold program (http://unafold.rna.albany.edu/?q=mfold ) was used to determine whether the miATXN3 candidates are folded correctly into their secondary structures. The complete sequences were ordered from GeneArt gene synthesis (Invitrogen). These constructs were subsequently cloned into an expression vector containing the cytomegalovirus (CMV) immediate-early enhancer fused to chicken β-actin (CAG) promoter (Inovio, Plymouth Meeting, PA, USA) using the EcoRV and BamHI sites. For generation of the Luc reporter, the complete ATXN3 mRNA (NM_004993.5) sequence was synthesized at GeneArt gene synthesis and cloned in the 3′ UTR of the RL gene of the psiCHECK-2 vector (Promega, Madison, WI, USA). The FL gene was also expressed in this vector and served as internal control.
3
Characterization of Igf2 P3 Promoter
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The 342-bp Igf2 P3 promoter sequence containing the murine Igf2 P3 proximal promoter [33 (link),34 (link)] was synthesized using GeneArt Gene Synthesis (Thermo Fisher Scientific). The transcription start site (TSS) sequence was localized using the Eukaryotic Promoter Database [35 (link)]. The Igf2 P3 promoter sequence was then cloned into the Nanoluciferase expressing promoterless vector pNL1.2[NlucP] (Promega) using NheI (5′) and EcoRV (3′) restriction sites with GeneArt Gene Synthesis. The resulting plasmid was called Igf2 P3 Nluc. Site-directed mutagenesis or promoter deletions were performed with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) with 500 nM of each forward and reverse primer (Table 1 ) and 50 ng of plasmid. PCR was performed at the following conditions: 98 °C for 30 s, followed by 25 cycles at 98 °C for 10 s, for 30 s at 65–71 °C (Table 1 ), and 72 °C for 150 s and ended at 72 °C for 2 min. The site-directed mutagenesis PCR product was visualized on 1% agarose gel and then treated with the KLD Enzyme Mix (New England Biolabs) prior to transformation into the E. coli strain DH5-α competent cells. Plasmids were extracted with NucleoSpin Plasmid Transfection-grade (Macherey-Nagel), and their concentrations were measured using Nanodrop ND-1000. All plasmids were sequenced using Sanger sequencing (Giga-Genomics).
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