Establishing Stable Cell Lines for Gene Knockdown

Specific shRNAs against LINC01106 (sh/LINC01106#1/2), FUS (sh/FUS#1/2), Gli1 (sh/Gli1#1/2), Gli2 (sh/Gli2#1/2), and their relative negative control (sh/ctrl), were obtained from Genechem (Shanghai, China), together with pcDNA3.1/Gli4, pcDNA3.1/FUS, pcDNA3.1/Gli1, pcDNA3.1/Gli2, and the pcDNA3.1 empty vector (negative control). MiRNA mimics and NC mimics, miR-449b-5p inhibitor and NC inhibitor were all synthesized by GenePharma (Shanghai, China). All above plasmids were constructed into the pLKO.1 vector (Addgene #10878), followed by lentiviral particle preparation as previously described^{26 (link)}. Thereafter, lentiviral particles containing above plasmids were separately transfected into LoVo or SW1116 cells for 48 h by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Finally, stably transfected cells were screened out via 2 μg/ml of puromycin treatment for additional 48 h.

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Guo K., Gong W., Wang Q., Gu G., Zheng T., Li Y., Li W., Fang M., Xie H., Yue C., Yang J, & Zhu Z. (2020). LINC01106 drives colorectal cancer growth and stemness through a positive feedback loop to regulate the Gli family factors. Cell Death & Disease, 11(10), 869.

Publication 2020

(sh/FUS#1/2 sh/ctrl pcDNA3.1 empty vector miR-449b-5p inhibitor pLKO.1 vector Lipofectamine 3000

Cells Lipofectamine Mirna Plasmids Puromycin Shrnas Vector

Corresponding Organization : Jiangsu Cancer Hospital

Other organizations : University of Science and Technology of China, Southeast University, Nanjing General Hospital of Nanjing Military Command, Jiangsu Vocational College of Medicine, Anhui Medical University

Top 5 similar protocols

Variable analysis

independent variables

ShRNAs against LINC01106 (sh/LINC01106#1/2)
ShRNAs against FUS (sh/FUS#1/2)
ShRNAs against Gli1 (sh/Gli1#1/2)
ShRNAs against Gli2 (sh/Gli2#1/2)
PcDNA3.1/Gli4
PcDNA3.1/FUS
PcDNA3.1/Gli1
PcDNA3.1/Gli2
MiRNA mimics
MiR-449b-5p inhibitor

dependent variables

Not explicitly mentioned

control variables

Relative negative control (sh/ctrl)
PcDNA3.1 empty vector
NC mimics
NC inhibitor

controls

Relative negative control (sh/ctrl)
PcDNA3.1 empty vector
NC mimics
NC inhibitor

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