Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes
Naushin L. Hindul,
Amarjot Jhita,
Daiana G. Oprea,
Tasnim Alamgir Hussain,
Oksana Gonchar,
Miguel Angel Muro Campillo,
Laura O'Regan,
Masato T. Kanemaki,
Andrew M. Fry,
Kouji Hirota,
Kayoko Tanaka
Affiliations
Naushin L. Hindul
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Amarjot Jhita
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Daiana G. Oprea
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Tasnim Alamgir Hussain
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Oksana Gonchar
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Miguel Angel Muro Campillo
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Laura O'Regan
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Masato T. Kanemaki
Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka 411-8540, Japan
Andrew M. Fry
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
Kouji Hirota
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo, 192-0397, Japan
Kayoko Tanaka
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, University Road, Leicester LE1 7RH, UK
The human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes.
