Technical Resources
- John Fetter, Andrey Samsonov, Nathan Zenser, Fan Zhang, Hongyi Zhang, Dmitry Malkov. Endogenous Gene Tagging, Chapter in Methods in Molecular Biology Volume 1239, pp 231-240, 2015.
- Alexandre Grassart, Aaron T. Cheng, Fan Zhang, Nathan Zenzer, Yongmei Feng, David M. Briner, Gregory D. Davis, Dmitry Malkov & David G. Drubin. Actin and dynamin2 dynamics and interplay during clathrin mediated endocytosis. J. Cell Biol. Vol. 205 No. 5 721–735, 2014.
- Andrey Samsonov, Nathan Zenser, Fan Zhang, Hongyi Zhang, John Fetter, Dmitry Malkov. Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors. PLoS ONE 8(7): e68391, 2013.
- Andrey Samsonov, Hongyi Zhang, Fan Zhang, Nathan Zenser, John Fetter, Dmitry Malkov. Novel Reporter Cell Lines For Detection Of Endogenous Pathway Activity In Live Cells. Biowire, Spring 2012, p.13-18.
HER2-GFP and EGFR-RFP in SKOV3 cells
SKOV3 cells that have the genomic HER2 gene endogenously tagged with GFP gene and the genomic EGFR gene - with RFP gene. The endogenous EGFR-RFP fusion protein translocates from the plasma membrane to the endosomes after stimulation with 100 ng/ml EGF while the endogenous HER2-GFP fusion protein stays at the membrane. Zinc finger nuclease (ZFN) technology was used to tag two genes (HER2 and EGFR) by inserting a fluorescent reporter sequence at the end of the coding sequence of each locus (a C-terminal fusion). DIC + epi-Fluorescence, 2 fps, 7 sec.
STAT1-GFP and RFP-STAT3 in A549 cells
Endogenous STAT1/STAT3 nuclear translocation upon simultaneous activation. A549 lung carcinoma cells expressing the endogenous STAT1 protein tagged with GFP at the C-terminus and the endogenous STAT3 protein tagged with RFP at the N-terminus. Zinc finger nuclease (ZFN) technology was used to tag two genes (STAT1 and STAT3) by a fluorescent reporter sequences. The cells were preincubated with 1 µM of Hoechst 33342 and imaged live before and after addition of 100 ng/mL IFN-γ and 100 ng/mL IL-6. DIC + epi-Fluorescence, 2 fps, 9 sec.
EGFR-GFP in A549 cells
A549 cells that have the genomic epidermal growth factor receptor (EGFR) gene endogenously tagged with GFP gene. The endogenous EGFR-GFP fusion protein translocates from the plasma membrane to the endosomes after stimulation with 100 ng/ml EGF. Zinc finger nuclease (ZFN) technology was used to tag EGFR gene by inserting a fluorescent reporter sequence at the end of the EGFR coding sequence (a C-terminal fusion). DIC + epi-Fluorescence, 6 fps, 16 sec.
BFP-lamin B1, GFP-α-tubulin 1b, and RFP-β-actin in triple tagged U2OS cells
Zinc finger nuclease (ZFN) technology was used to tag three cytoskeletal genes by inserting a fluorescent reporter sequence behind the start codon of each locus. The integration resulted in endogenous expression of the corresponding fusion proteins. The following loci were tagged: LMNB1 (lamin B1, nuclear envelope), TUBA1B (α-tubulin 1b, microtubules), ACTB (β-actin, actin stress fibers) by BFP, GFP and RFP respectively in the same cells (U2OS). Differential interference contrast (DIC) and fluorescence microscopy images of an isolated triple knock-in clone are shown. The cells were imaged live in Hanks balanced salt solution supplemented with 2% fetal bovine serum using corresponding filter sets and 40x/1.3 oil objective. The bar is 25 μm. 7 fps, 5 sec.
Paclitaxel effect on RFP-α-tubulin in MCF10A cells
Paclitaxel (or Taxol -- cancer drug) effect on microtubules. MCF10A cells with a cytoskeletal protein α-tubulin RFP tagged. Zinc finger nuclease (ZFN) technology was used to tag TUBA1B gene by inserting a fluorescent reporter sequence behind the start codon of this locus. DIC + epi-Fluorescence, 10 fps, 22 sec.
GFP-lamin-B1 in U2OS - cell division
U2OS cell division. A protein that forms the inner nuclear lining, lamin B1, is GFP tagged. Zinc finger nuclease (ZFN) technology was used to tag LMNB1 gene by inserting a fluorescent reporter sequence behind the start codon of this locus. DIC + epi-Fluorescence, 10 fps, 18 sec.
GFP-based biosensor detects endogenous EGF receptor activity in A549 cells
Detection of endogenous EGFR phosphorylation and internalization by a GFP-based biosensor. EGF causes the biosensor to translocate towards the plasma membrane and bind EGFR followed by internalization in A549 cells. The nucleus was labeled with DRAQ5. DIC + epi-Fluorescence, 6 fps, 12 sec.
GFP-β-actin in U2OS cells
Lamellipodia formation by actin polymerization. U2OS cells with a cytoskeletal protein β-actin GFP tagged. Zinc finger nuclease (ZFN) technology was used to tag ACTB gene by inserting a fluorescent reporter sequence behind the start codon of this locus. DIC + epi-Fluorescence, 10 fps, 19 sec.
HMGA1-GFP in U2OS - cell division
U2OS cell division. A non-histone DNA binding protein HMGA1 is GFP tagged. Zinc finger nuclease (ZFN) technology was used to tag HMGA1 gene by inserting a fluorescent reporter sequence in front of the stop codon of this locus. DIC + epi-Fluorescence, 6fps, 19 sec.
GFP-lamin-B1 and RFP-β-actin
Actin supported ruffles. Dual tagged U2OS cells: GFP-lamin B1 (nuclear envelope), RFP-β-actin (actin stress fibers). Zinc finger nuclease (ZFN) technology was used to tag two genes (LMNB1 and ACTB) by inserting a fluorescent reporter sequence behind the start codon of each locus. DIC + epi-Fluorescence, 10fps, 17 sec.
