A Cell Competition System with One Gene Expression from a Single-copy Gene in One Cell
Yoshinori Hasegawa, Megumi Nakano, Tsutomu Hosouchi, Takashi Watanabe, Izumi Yamaguchi, Manabu Nakayama, Osamu Ohara.
Abstract
Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared.
Introduction
Cell competition (CC) is a biological mechanism highly conserved from Drosophila to vertebrates and results in the elimination of less fit cells by their more fit neighbors. CC is also involved in the interactions between tumor and neighbor cells, as they are crucial to the initial events of tissue transformation and subsequent tumor outgrowth and invasion. In particular, overexpression or activation of an oncogene frequently leads to uncontrolled cell growth or growth arrest.
Materials and Methods:
All analyses were performed using the CLC genomics workbench version 23 (QIAGEN, Hilden, Germany) with the default settings. Using the”Trim Reads” tool, adapter sequences were removed from the raw reads, and base trimming was conducted. Reads shorter than 25 bp were removed prior to mapping. Each read was mapped to the human genome hg38 using the”RNA-Seq Analysis” tool.
Discussion
To accurately compare the effects of transgenes in inserted cells, it is necessary to express all genes at the same level. However, this is challenging to achieve with gene expression methods using plasmids, transposons, and virus-based vectors as expression from the same copies of transgenes in all transfected cells is desired. In addition, for studying CC, it is necessary to insert multiple genes simultaneously. In this study, we utilized KDRI HaloTag 48 NR clones to achieve expression of a single-copy gene within individual cells. We then examined the effects of elevated NR expression levels on the cell proliferation rate in immortalized human embryonic kidney (HEK293) cells.
Citation: Hasegawa Y, Nakano M, Hosouchi T, Watanabe T, Yamaguchi I, Nakayama M, et al. (2024) A cell competition system with one gene expression from a single-copy gene in one cell. PLoS ONE 19(7): e0302451. https://doi.org/10.1371/journal.pone.0302451
Editor: Chien-Feng Li, National Institute of Cancer Research, TAIWAN
Received: January 11, 2024; Accepted: April 3, 2024; Published: July 5, 2024.
Copyright: © 2024 Hasegawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw data were deposited in the DNA Data Bank of Japan (DDBJ;) (DDBJ; accession numbers DRA017470, PRJDB17053).
Funding: JSPS KAKENHI Grant Number JP19K05178 [to Y.H.] Himawari Venture Development Fund 2016 [to KGT Inc.].
Competing interests: The authors have declared that no competing interests exist.
