The cDNAs of human HCN4, human wild type (SGO1-WT, NM_001199252.3) and mutant (SGO1-K23E) SGO1 were subcloned into the lentiviral vector pLentiCMV in which the GFP coding section was deleted from the backbone of pLentiCMV-GFP-Neo (Addgene #17447, gift from Eric Campeau & Paul Kaufman). The GFP was cloned into the N-terminal of HCN4 and mCherry was cloned into the N-terminal of SGO1-WT and SGO1-K23E to form fusion proteins. The lentiviral plasmids carrying SGO1-WT, SGO1-K23E, HCN4, SGO1-WT-mCherry, SGO-K23E-mCherry, or HCN4-GFP were co-transfected into HEK293T cells with packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260), which were gifts from Didier Trono. The supernatants containing virus were harvested 48 and 72 h after transfection and concentrated using Lenti-XTM concentrator (TaKaRa Bio). The viral pellet was resuspended in serum-free DMEM medium and viral titers were measured with QuickTiter Lentivirus Quantitation Kit (Cell Biolabs). Viral stocks were stored at –80 °C until use.
Plenticmv gfp neo
Manufactured by Addgene
The PLentiCMV-GFP-Neo is a lentiviral vector that contains the GFP reporter gene and a neomycin resistance gene. It is designed for the stable transduction and expression of genes in mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Quicktiter lentivirus quantitation kit, by Cell Biolabs (1 mentions)
Lenti xtm concentrator, by Takara Bio (1 mentions)
Pspax2, by Addgene (1 mentions)
In fusion kit, by Takara Bio (1 mentions)
Pcdh cmv mcherry t2a puro, by Addgene (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Tissue freezing medium, by Leica (1 mentions)
Cm1950, by Leica (1 mentions)
4 protocols using plenticmv gfp neo
1
Lentiviral Vector Construction and Quantification
2
Generation of Claudin-Deficient Cell Lines
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pLenti CMV GFP Neo (Addgene Plasmid #17447) was used for the construction of expression vectors. DNA fragments encoding mouse claudin-3, claudin-3 lacking the COOH terminus KDYV (delYV), and claudin-3 carrying C113S, C116S, C192S, and C194S mutations (4S) were subcloned into the pLenti CMV GFP Neo vector. Claudin-null cells were generated by using the CRSPR-Cpf1 system. The target sequences were as follows:
claudin-1 (mouse), 5′-ATCCTGGCTTCTCTGGGATGGAT-3′; claudin-3 (mouse), 5′-GGCCTTCATCGGCAGCAGCATCA-3′; claudin-4 (mouse), 5′-CCTCTTCTGCCCGGAAGCCACCA-3′; claudin-7 (mouse), 5′-GCAGCTCATCATGCCGGTGCTCT-3′; claudin-9 (mouse), 5′-GTGTCCTGTGCCCTGCCACTGTG-3′; and claudin-10b (mouse), 5′-GCCAACCTGTGGAAGATCTGCGT-3′.
claudin-1 (mouse), 5′-ATCCTGGCTTCTCTGGGATGGAT-3′; claudin-3 (mouse), 5′-GGCCTTCATCGGCAGCAGCATCA-3′; claudin-4 (mouse), 5′-CCTCTTCTGCCCGGAAGCCACCA-3′; claudin-7 (mouse), 5′-GCAGCTCATCATGCCGGTGCTCT-3′; claudin-9 (mouse), 5′-GTGTCCTGTGCCCTGCCACTGTG-3′; and claudin-10b (mouse), 5′-GCCAACCTGTGGAAGATCTGCGT-3′.
3
Cloning and Engineering of Tagged NELFB
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Cloning was performed using In-Fusion kit (Takara, 638,947). C-terminal 3× flag-tagged mouse NELFB was introduced into the pLenti-CMV-GFP-neo (Addgene: 17447) backbone. NELFB truncations and site-specific point mutagenesis were generated using PrimeSTAR GXL DNA Polymerase (Takara, R050B) and different sets of primers (Tables S1 and S10 ). OsTIR1 was cloned into pCDH-CMV-mcherry-T2A-puro (Addgene: 72264). C-terminal mini-AID–tagged mouse NELFB and human NELFC were cloned into pLenti-6.3/V5-DEST-GFP (Addgene: 40125). C-terminal AID–tagged human NELFB with silent mutation resistant to single guide RNA (sgRNA) was cloned into pLenti-6.3/V5-DEST-GFP. APEX2 was cloned to the C-terminal of WT and MTs (MT144 and MT194) NELFB into pLenti-CMV-GFP-neo backbone (Table S8 ). Sequences of all constructs were verified by sequencing.
4
Transplantation of Genetically Modified Neural Stem Cells
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miNSCs and hiNSCs infected with the pLenti-CMV-GFP-Neo (Addgene, 17447) lentiviruses were cultured on a 10 cm tissue culture dish in the NSC medium in the presence of G418 (500 μg/ml). After 48 h in culture, GFP-tagged miNSCs survived and produced small neurospheres. These neurospheres were digested with Accutase and centrifuged, and the dissociated cells were resuspended and diluted to a final concentration of 1 × 105 cells/μl. Two microliters of the cell suspension were injected into the hippocampal region (anterior-posterior, 2 mm; left–right lateral, 1.5 mm; and dorso-ventral, 2 mm) of an anesthetized C57BL/6 J mouse (8 weeks old, male or female) using a stereotaxic apparatus (RWD Life Science). The injected mouse was placed on an electric blanket until it fully awoke.
One to 1.5 months after transplantation, the animals were anesthetized and perfused by cardiac puncture with PBS followed by 4% paraformaldehyde. The brains were collected, fixed with 4% paraformaldehyde for 4 h at 4 °C with slow shaking and dehydrated in 30% sucrose for 48 h. They were then encased in tissue freezing medium (Leica) and stored at − 80 °C, followed by sectioning into 20 μm coronal sections using a cryostat (CM1950, Leica) for immunohistochemistry.
One to 1.5 months after transplantation, the animals were anesthetized and perfused by cardiac puncture with PBS followed by 4% paraformaldehyde. The brains were collected, fixed with 4% paraformaldehyde for 4 h at 4 °C with slow shaking and dehydrated in 30% sucrose for 48 h. They were then encased in tissue freezing medium (Leica) and stored at − 80 °C, followed by sectioning into 20 μm coronal sections using a cryostat (CM1950, Leica) for immunohistochemistry.
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