Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Knockdown of Atg5 expression by lentivirus-delivered shRNA

JL Jhao-Yin Lin
HH Hsing-I Huang
request Request a Protocol
ask Ask a question
Favorite

Lentivirus-based shRNA constructs targeting human ATG5 were obtained from the National RNAi Core Facility, Taiwan, and a negative control construct (shNC) was obtained from Dr. Shih Shin-Ru. To prepare the lentivirus, 293 T cells were transfected with pLKO-shRNA and two helper plasmids, pMD. G and pCMVΔ8.91, with Mirus transfection reagent. After 48 h, the supernatant was collected and stored at − 80 °C. To generate a stable ATG5-knockdown IMR-32 clone, lentivirus expressing shATG5 was used to transfect IMR-32 cells, and puromycin (1 μg/ml) was used to select cells expressing shATG5.

Copyright and License information: The Author(s) ©2020
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A