The 47S rDNA gene promotor sequence was custom-made by Genecust, containing nucleotides -1000 up to +60 from the human 47S rDNA transcription start site of the 47S rDNA gene sequence (Ensembl), and cloned into the pNL1.2 vector (Promega, Madison, Wisconsin, USA). The pNL1.2_47S-rDNA promoter plasmid was transfected into SW1353 cells (30.000 cells/cm2; n = 6 samples per condition) with Fugene6. Five hours Post-transfection, cells were incubated for 24 hours with 1 nM BMP7. Post-stimulation, cells were harvested for bioluminescence analysis using cell culture lysis buffer (Promega). Promotor-activity was measured with the Nano-Glo Luciferase Assay System (Promega) on a Tristar LB942 (Berthold Technologies, Bad Wildbad, Germany). Relative differences were determined as compared to control conditions following correction for background and normalization by DNA-content. DNA-content was measured using a SYBR-GREEN assay (Invitrogen).
Pnl1.2 vector
Manufactured by Promega
Sourced in United States
The PNL1.2 vector is a plasmid designed for gene expression and cloning. It contains a multiple cloning site, a T7 promoter, and a kanamycin resistance gene. The core function of the PNL1.2 vector is to serve as a tool for molecular biology research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy vector, by Promega (2 mentions)
Tristar lb942, by Berthold Technologies (1 mentions)
Sybr green assay, by Thermo Fisher Scientific (1 mentions)
Cell culture lysis buffer, by Promega (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Plasmid maxi kit, by Qiagen (1 mentions)
In fusion hd cloning plus kit, by Takara Bio (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Phusion dna polymerase, by Zymo Research (1 mentions)
5 protocols using pnl1.2 vector
1
47S rDNA Promoter Activity Regulation by BMP7
2
Constructing cNANOG Luciferase Vectors
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To construct NanoLuc luciferase expression vectors, the 5′ flanking region of the cNANOG gene was amplified using genomic DNA extracted from adult chicken blood and inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA). Primer sets were used to clone differently sized fragments of the cNANOG promoter (Table 1 ). Then, different lengths of the 5′ upstream region of the cNANOG gene were inserted between the KpnI and XhoI sites of the pNL1.2 vector (Promega).
List of primer sequences used to clone the NANOG promoter
| Primer name | Primer sequence (5′ → 3′) |
|---|---|
| cNANOG − 3550 bp_F | AAGCTTTGTCCTTTTCTTGACC |
| cNANOG − 3375 bp_F | CTGGAGTCAAGGGCTGTGG |
| cNANOG − 3154 bp_F | TGGGCCCCTCGTTACAGCT |
| cNANOG − 2928 bp_F | CCAGCAGTACAAGCTCCGAA |
| cNANOG − 1988 bp_F | GCGACACGTGGAACA |
| cNANOG − 945 bp_F | CATGGGGGTGTCTGCTC |
| cNANOG − 627 bp_F | CTTCTTTGTGCTCCTCC |
| cNANOG − 442 bp_F | CTGCAGTCTGCAATGC |
| cNANOG − 407 bp_F | AATGTCCCGGGGGGGTCTCTGG |
| cNANOG − 377 bp_F | CCATTCTTTGTACTTGGGTGGGGACCGATGAG |
| cNANOG − 312 bp_F | CGAGGGCGGGGGTGCCAGCCCAG |
| cNANOG − 250 bp_F | CTGCAGTCTGCTCCTCC |
| cNANOG − 210 bp_F | CTGCAGTCTGCAATGC |
| cNANOG − 170 bp_F | CCAAAGGGGGAAGCTGC |
| cNANOG − 130 bp_F | ACTCTCCGAATATCCCCATAGC |
| cNANOG − 69 bp_F | TCGTGACAATCTCTTG |
| cNANOG promoter_R | GGTCGGGACGACACCT |
3
Lentiviral Transcription Factor Reporters
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Binding sequences of 11 specific transcription factor complexes (Supplementary Table 2) in front of a minimal promoter (sequence: AGAGGGTATATAATGGAAGCTCGACTTCCAG) were custom synthesized by Genecust (Boynes, France). The promoter sequences were directionally cloned into the pNL1.2 vector (Promega) through enzymatic restriction and ligation with T4 DNA Ligase (NEB). Subsequently, lentiviral constructs were generated by re-cloning pNL1.2 reporters with the In-Fusion HD Cloning Plus kit (TakaraBio) into the ClaI-linearized pLVX-EF1α-IRES-Puro transfer vector (TakaraBio) according to the manufacturer's instructions (primers; Supplementary Table 3). Plasmid isolation was performed with Plasmid Maxi Kit (Qiagen).
4
PARK2 Gene Targeting Donor Construct
5
Constructing NanoLuc Luciferase Vectors
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To construct NanoLuc luciferase expression vectors, the 5' anking region of the cNANOG gene was ampli ed using genomic DNA extracted from adult chicken blood and inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA). Primer sets were used to clone differently sized fragments of the cNANOG promoter (Table 1). Then, different lengths of the 5' upstream region of the cNANOG gene were inserted between the KpnI and XhoI sites of the pNL1.2 vector (Promega).
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