Inducible Expression of KCTD17 and Trichoplein in RPE1 Cells

Tet-On RPE1 cell lines that expressed myc-KCTD17 or MBP-trichoplein-flag (WT or K50/57R) were established with the same procedure described previously29 (link)46 (link)47 (link). The rtTA-advanced segment and the tTS transcriptional silencer segment from pTet-On Advanced and pQC-tTS-IN (BD Clontech) were recombined into the retroviral vector pDEST-PQCXIP and pDEST-PQCXIN, respectively, by the LR reaction (Invitrogen) to generate PQCXIN-Tet-On ADV and PQCXIP-tTS. The Elongation factor 1 alpha promoter (EF) in CSII-EF-MCS (a gift from Hiroyuki Miyoshi, RIKEN BioResource Center, Tsukuba, Japan) was replaced with a Tet-responsive promoter (TRE-Tight) from pTRE-Tight (BD Clontech) followed by a modified RfA fragment (Invitrogen) to make a Tet-responsive lentivirus vector, CSII-TRE-Tight-RfA. Fusion cDNAs with siRNA-resistant KCTD17 and trichoplein were recombined into the lentiviral vector by the LR reaction (Invitrogen) to generate CSII-TRE-Tight-myc-KCTD17 and CSII-TRE-Tight-MBP-trichoplein-3xFLAG, respectively. For induction of myc-KCTD17 or MBP-trichoplein-flag, Tet-On RPE1 cells were treated with 30 or 100 ng ml⁻¹ doxycycline (Sigma-Aldrich), respectively.

Free full text: Click here

Kasahara K., Kawakami Y., Kiyono T., Yonemura S., Kawamura Y., Era S., Matsuzaki F., Goshima N, & Inagaki M. (2014). Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nature Communications, 5, 5081.

Publication 2014

LR reaction TRE-Tight pTRE-Tight doxycycline

Cdnas Cell lines Cells Doxycycline Elongation factor 1 alpha Lentivirus Retroviral Sirna Transcriptional Vector

Corresponding Organization : Nagoya University

Other organizations : Aichi Cancer Center, National Institute of Advanced Industrial Science and Technology, RIKEN Center for Computational Science, Japan Biological Informatics Consortium

Top 5 similar protocols

Variable analysis

independent variables

Expression of myc-KCTD17 or MBP-trichoplein-flag (WT or K50/57R) in Tet-On RPE1 cell lines

dependent variables

Not explicitly mentioned

control variables

Tet-On RPE1 cell lines
Procedure for establishing the cell lines as described previously in reference 29

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!