Dual-reporter FUCCI system monitors cell cycle

C4-2B4 cells were sequentially transduced with two retroviral vectors, one marking G1 and other S-G2M cell cycle phases. Cells were first transduced with pRetroX-G1-Red G1 reporter containing DNA replication factor 1 (CDT) fused with mCherry and selected with puromycin for 7 days [20 (link)]. Cells were subsequently transduced with pRetroX-SG2M reporter containing Geminin fused with Cyan and selected with neomycin plus puromycin for 7 days. CDT-mCherry levels peak in G1 but drop after S as CDT-mCherry is degraded by SCF E3 ubiquitin ligase. Geminin-Cyan levels are high in S-G2M but drop in G1 as Geminin-Cyan is degraded by APC ubiquitin ligase. The dynamic shift of C4-2B4-FUCCI cells from G1 (red) into S-G2M (green) was monitored live at the single cell level in a BioStation (Nikon) [48 (link)].

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Pan T., Lin S.C., Lee Y.C., Yu G., Song J.H., Pan J., Titus M., Satcher R.L., Panaretakis T., Logothetis C., Yu-Lee L.Y, & Lin S.H. (2021). Statins reduce castration-induced bone marrow adiposity and prostate cancer progression in bone. Oncogene, 40(27), 4592-4603.

Publication 2021

BioStation

Cell Cell cycle Dna replication E3 ubiquitin ligase Factor 1 Geminin Ligase Neomycin Puromycin Retroviral Ubiquitin Vectors

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, Baylor College of Medicine

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Variable analysis

independent variables

Transduction with pRetroX-G1-Red G1 reporter containing DNA replication factor 1 (CDT) fused with mCherry and selected with puromycin for 7 days
Transduction with pRetroX-SG2M reporter containing Geminin fused with Cyan and selected with neomycin plus puromycin for 7 days

dependent variables

Dynamic shift of C4-2B4-FUCCI cells from G1 (red) into S-G2M (green) monitored live at the single cell level

control variables

Not explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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