Lentiviral Transduction for GCNT1 Modulation

Cell culture and the cell lines used were as described previously^{34 (link)}. The stable cell lines used in the study were created using lentiviral transduction. For knockdown of GCNT1 the following shRNA lentiviral particles were purchased from Santa Cruz (GCNT1 shRNA sc-92945-V and Control shRNA sc-108080). Transductions were carried out according to the manufacturer’s instructions using an MOI of 5. For overexpression of GCNT1 the following Lentifect Purified lentiviral particles were purchased from Tebu-Bio (GCNT1 LPP-Z6088-Lv242-050-S and Negative Control 217LPP-NEG-Lv242-025-C). Transductions were carried out according to the manufacturer’s instructions using an MOI of 5. For the metastasis study, PC3 cells were transduced with Firefly Luciferase Lentivirus (BPS Bioscience, 79692-H) at a MOI of 1 in media containing 5 µg/ml polybrene. Stable cell lines were selected with 200 μg/ml hygromycin, and then further selected for GCNT1 overexpression using the methods described above.

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Hodgson K., Orozco-Moreno M., Scott E., Garnham R., Livermore K., Thomas H., Zhou Y., He J., Bermudez A., Garcia Marques F.J., Bastian K., Hysenaj G., Archer Goode E., Heer R., Pitteri S., Wang N., Elliott D.J, & Munkley J. (2023). The role of GCNT1 mediated O-glycosylation in aggressive prostate cancer. Scientific Reports, 13, 17031.

Publication 2023

Control shRNA sc-108080 Firefly Luciferase Lentivirus

Cell culture Cell lines Firefly luciferase Hygromycin Lentivirus Metastasis Pc3 cells Polybrene Shrna

Corresponding Organization :

Other organizations : Newcastle University, University of Sheffield, Stanford University

Top 5 similar protocols

Variable analysis

independent variables

Knockdown of GCNT1 using shRNA lentiviral particles
Overexpression of GCNT1 using Lentifect Purified lentiviral particles

dependent variables

Metastasis of PC3 cells transduced with Firefly Luciferase Lentivirus

control variables

Cell culture and cell lines as described previously
Transductions carried out according to manufacturer's instructions using an MOI of 5
Stable cell lines selected with 200 μg/ml hygromycin

positive controls

Control shRNA sc-108080
Negative Control 217LPP-NEG-Lv242-025-C

negative controls

Control shRNA sc-108080
Negative Control 217LPP-NEG-Lv242-025-C

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