scholarly journals Temporal-Spatial Visualization of Endogenous Chromosome Rearrangements in Living Cells

2019 ◽  
Author(s):  
Haifeng Wang ◽  
Muneaki Nakamura ◽  
Dehua Zhao ◽  
Cindy M Nguyen ◽  
Cordelia Yu ◽  
...  
Keyword(s):  
Real Time ◽  
Gene Editing ◽  
Genome Rearrangement ◽  
Cell Types ◽  
Living Cells ◽  
Primary Cells ◽  
Chromosomal Disorders ◽  
Time Dynamics ◽  
Chromosome Translocations ◽  
Patau Syndrome

AbstractVisualizing the real-time dynamics of genome rearrangement in single living cells is core to studying genomics and diagnostics. Here, we report a robust, versatile approach named CRISPR Live-cell fluorescent in situhybridization (LiveFISH) for multi-locus genome tracking and cytogenetic detection in a broad variety of cell types including primary cells. LiveFISH utilizes an intrinsic stability switch of CRISPR guide RNAs, which enables efficient and accurate detection of chromosomal disorders such as Patau Syndrome in prenatal amniotic fluid cells and allows multi-locus tracking in human T lymphocytes. Using LiveFISH, we are able to detect and track real-time spatiotemporal dynamics of non-homologous endogenous chromosome translocations induced by gene editing. This new approach enables FISH imaging in living primary cells, which can provide useful insights into the spatiotemporal changes of genome organization and rearrangements in normal and diseased primary cells and will enable fast cytogenetic visualization of various gene-editing associated chromosomal translocations.

CRISPR-mediated live imaging of genome editing and transcription

Science ◽  
2019 ◽  
Vol 365 (6459) ◽  
pp. 1301-1305 ◽  
Author(s):  
Haifeng Wang ◽  
Muneaki Nakamura ◽  
Timothy R. Abbott ◽  
Dehua Zhao ◽  
Kaiwen Luo ◽  
...  
Keyword(s):  
Real Time ◽  
Genome Editing ◽  
Single Cells ◽  
Cell Types ◽  
Live Imaging ◽  
Dna Double Strand Breaks ◽  
Chromosomal Disorders ◽  
Dna And Rna ◽  
Multiple Loci ◽  
Patau Syndrome

We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9–mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.

scholarly journals Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3042
Author(s):  
Eun Ju Lee ◽  
Khurshid Ahmad ◽  
Shiva Pathak ◽  
SunJu Lee ◽  
Mohammad Hassan Baig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Cell Adhesion ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Primary Cells ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Promote Cell Adhesion

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

A multicolor DNA tetrahedron nanoprobe for analyzing human telomerase in living cells

2021 ◽  
Author(s):  
Ruiyuan Zhang ◽  
Ruixue Zhang ◽  
Wei Jiang ◽  
Xiaowen Xu
Keyword(s):  
Real Time ◽  
Length Distribution ◽  
Telomerase Activity ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Dna Tetrahedron ◽  
Human Telomerase

A sequentially lighting-up multicolor DNA tetrahedron nanoprobe is constructed for imaging telomerase activity, real-time monitoring telomerase action and determining product length distribution in living cells.

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