To generate knockdown PRPK cells, pLKO.1-shPRPK or pLKO.1-mock lentivirus plasmids were co-transfected with psPAX2 and pMD2.0G into HEK 293T cells using the jetPEI poly transfection reagent (Polyplus-transfection SA; New York, NY). The human shPRPK full hairpin sequence is #1, 5′-CCGGGAGATTATATTCAGTCCACTACTCGAGTAGTGGACTGAATATAATCTCTTTTTG-3′, and #2, 5′-CCGGCATAGACTTTGGGCTGAGTTTCTCGAGAAACTCAGCCCAAAGTCTATGTTTTTG-3′. The viral particles containing 8 μg/mL polybrene were infected into A431 cells overnight and the medium was replaced with fresh complete growth medium. After 24 h, A431 cells were selected with 2 μg/mL of puromycin. The shMock or shPRPK-transfected A431 cells were incubated for 10 days as described previously (53 (link)). Colony formation was observed and analyzed using a LEICA DM IRB microscope (Nikon Corporation) and the ImagePro Plus software (v.6.1) program (Media Cybernetics Inc., Rockville, MD).
Jetpei poly transfection reagent
Manufactured by Polyplus Transfection
JetPEI is a cationic polymer-based transfection reagent designed to facilitate the delivery of nucleic acids, such as plasmid DNA or siRNA, into mammalian cells. It forms compact complexes with the genetic material, enabling its efficient uptake by the target cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using jetpei poly transfection reagent
1
Generating PRPK Knockdown Cells
2
Generating PRPK Knockdown Cells
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To generate knockdown PRPK cells, pLKO.1-shPRPK or pLKO.1-mock lentivirus plasmids were co-transfected with psPAX2 and pMD2.0G into HEK 293T cells using the jetPEI poly transfection reagent (Polyplus-transfection SA; New York, NY). The human shPRPK full hairpin sequence is #1, 5′-CCGGGAGATTATATTCAGTCCACTACTCGAGTAGTGGACTGAATATAATCTCTTTTTG-3′, and #2, 5′-CCGGCATAGACTTTGGGCTGAGTTTCTCGAGAAACTCAGCCCAAAGTCTATGTTTTTG-3′. The viral particles containing 8 μg/mL polybrene were infected into A431 cells overnight and the medium was replaced with fresh complete growth medium. After 24 h, A431 cells were selected with 2 μg/mL of puromycin. The shMock or shPRPK-transfected A431 cells were incubated for 10 days as described previously (53 (link)). Colony formation was observed and analyzed using a LEICA DM IRB microscope (Nikon Corporation) and the ImagePro Plus software (v.6.1) program (Media Cybernetics Inc., Rockville, MD).
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