The RANKL specific gRNA construct (pNV-RANKL/KO) is based on the parenteral gRNA-Cas9-2A-GFP vector, which is transiently expressing gRNA and Cas9 and enables GFP selection of transfected MSCs, was purchased from abm (Richmond, Canada). Three vectors were purchased with the following gRNA sequences: pNV-RANKL1/KO—5’-CAGGAATTACAACATATCGT-3’ (472221110290), pNV-RANKL2/KO—5’-CAGCGATGGTGGATGGCTCA-3’ (472221110390), pNV-RANKL3/KO—5’-TTAATAGTGAGATGAGCAAA-3’ (472221110490). pmaxGFP (purchased from Lonza, Human MSC Nucleofector® Kit) was used to control transfection efficiency. Plasmids were propagated by transforming Escherichia Coli (Mix&Go! Competent Cells—DH5 Alpha, Zymo Research) using standard procedures. The plasmids were purified using the ZymoPURE™ II Plasmid Midiprep Kit (Zymo Research) according to the manufacturer’s instructions. The plasmid concentration was measured with the NanoDrop™ One. The plasmids were stored at—20°C.
Cas9 2a gfp vector
Manufactured by Applied Biological Materials
Sourced in Canada
The Cas9-2A-GFP vector is a plasmid that expresses the Cas9 protein and the green fluorescent protein (GFP) reporter. The Cas9 protein is a key component of the CRISPR-Cas9 gene editing system, which is used for targeted genome modification. The 2A peptide sequence allows for the co-expression of Cas9 and GFP from a single open reading frame, enabling the identification of successfully transfected cells.
Automatically generated - may contain errors
2 protocols using cas9 2a gfp vector
1
RANKL Knockout gRNA Constructs
2
RANKL Knockout Vector Construction
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The RANKL specific gRNA construct (pNV-RANKL/KO) based on the parenteral gRNA-Cas9-2A-GFP vector, which is transiently expressing gRNA and Cas9 and enables GFP selection of transfected MSCs, was purchased from abm (Richmond, Canada). Three vectors were purchased with the following gRNA sequences:
pNV-RANKL1/KO -5'-CAGGAATTACAACATATCGT-3' (472221110290), pNV-RANKL2/KO -5'-CAGCGATGGTGGATGGCTCA-3' (472221110390), pNV-RANKL3/KO -5'-TTAATAGTGAGATGAGCAAA-3' (472221110490). pmaxGFP (purchased from Lonza, Human MSC Nucleofector® Kit) was used to control transfection efficiency. Plasmids were propagated by transforming Escherichia Coli (Mix&Go! Competent Cells -DH5 Alpha, Zymo Research) using standard procedures.
The plasmids were purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research) according to the manufacturer's instructions. The plasmid concentration was measured with the NanoDrop One. The plasmids were stored at -20°C.
pNV-RANKL1/KO -5'-CAGGAATTACAACATATCGT-3' (472221110290), pNV-RANKL2/KO -5'-CAGCGATGGTGGATGGCTCA-3' (472221110390), pNV-RANKL3/KO -5'-TTAATAGTGAGATGAGCAAA-3' (472221110490). pmaxGFP (purchased from Lonza, Human MSC Nucleofector® Kit) was used to control transfection efficiency. Plasmids were propagated by transforming Escherichia Coli (Mix&Go! Competent Cells -DH5 Alpha, Zymo Research) using standard procedures.
The plasmids were purified using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research) according to the manufacturer's instructions. The plasmid concentration was measured with the NanoDrop One. The plasmids were stored at -20°C.
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