Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Pri-miRNA overexpression system

MZ Meng-Jie Zhao
JX Jun Xie
WS Wen-Jie Shu
HW Hong-Yan Wang
JB Jianping Bi
WJ Wei Jiang
HD Hai-Ning Du
ask Ask a question
Favorite

Pri-miRNA sequences were searched from UCSC Genome Browser (http://genome.ucsc.edu) and a nucleotide segment containing mi-15b or mi-322 was cloned into pHAGE-CMV vector using the restriction enzymes NotI and XhoI (New England Biolabs). The primers used for construction of pri-miRNA are as follows: pri-miR-15b forward (F): 5′-ATAAGAATGCGGCCGCGCCACCGGCATTG-ACTTAGACCATAATC-3′; pri-miR-15b reverse (R): 5′-CCGCTCGAGCACTACGCCAATATTTACGTG- 3′; pri-miR-322 forward (F): 5′-ATAAGAATGCGGCCGCGCCACCCTGAGGTAAGAGTCTCCTCC-3′; pri-miR-322 reverse (R): 5′-CCGCTCGAGGTGACCCTCACTAGACTAA-G-3′. 293 T cells were infected and selected according to the lentiviral expression and packaging protocol described above. The packaged virus was used to infect C2C12 cells to generate pri-miRNA stably expressed cell lines.

Copyright and License information: The Author(s) ©2019
Open Access This 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