To design IGFBP-3 shRNA construct, the rat IGFBP-3 gene sequence (GI:M31837) was analyzed for a potential siRNA target using the web-based siRNA target finder and design tool provided on the Ambion website (Ambion, Inc., Austin, TX). Double-stranded siRNAs (nucleotide position: 611) were transcribed “in vitro” using the Silencer siRNA construction kit (Ambion) following the manufacturer’s instructions. The inhibitory siRNA (5′-GCGCTACAAAGTTGACTATGA-3′, nucleotide position 611) was then cloned into the pGPU6/GFP/Neo plasmid vector (2nd version, Ambion) as a short hairpin DNA sequence (5′ sense strand:5′-CACCGCGCTACAAAGTTGACTATGATTCAAGAGATCATAGTCAACTTTGTAGCGCTTTTTTG-3′) according to the manufacturer’s instructions. The pGPU6/GFP/Neo-IGFBP-3 shRNA plasmid was purified using Endo-free Maxi kit (Qiagen, Valencia, CA). Plasmids were quantitated by spectrophotometry and prepared in 0.9% saline solution at a concentration of 1.0μg/μL.
Pgpu6 gfp neo plasmid vector
Manufactured by Thermo Fisher Scientific
The PGPU6/GFP/Neo plasmid vector is a DNA construct used for genetic engineering applications. It contains a GFP (Green Fluorescent Protein) gene and a neomycin resistance gene, allowing for selection of successfully transfected cells. The vector utilizes the U6 promoter for gene expression.
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Lab products found in correlation
2 protocols using pgpu6 gfp neo plasmid vector
1
Generating IGFBP-3 Silencing Construct
2
Constructing IGFBP-3 shRNA Plasmid
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To design IGFBP-3 shRNA construct, the rat IGFBP-3 gene sequence (GI: M31837) was analyzed for a potential siRNA target using the web-based siRNA target finder and design tool provided on the Ambion website (Ambion, Inc., Austin, TX). Double-stranded siRNAs (nucleotide position: 611) were transcribed in vitro using the Silencer siRNA construction kit (Ambion) following the manufacturer’s instructions. The inhibitory siRNA (5′-GCGCTACAAAGTTGACTATGA-3′, nucleotide position 611) was then cloned into the pGPU6/GFP/Neo plasmid vector (2nd version, Ambion) as a short hairpin DNA sequence (5′ sense strand: 5′-CACCGCGCTACAAAGTTGACTATGATT CAAGAGATCATAGTCAACTTTGTAGCGCTTTTTTG-3′) according to the manufacturer’s instructions. The pGPU6/GFP/Neo- IGFBP-3 shRNA plasmid was purified using the Endo-free Maxi kit (Qiagen, Valencia, CA). Plasmids were quantitated by spectrophotometry and prepared in 0.9% saline solution at a concentration of 1.0 μg/μL.
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