scholarly journals Knock-in of labeled proteins into 5’UTR enables highly efficient generation of stable cell lines

2020 ◽  
Author(s):  
Faryal Ijaz ◽  
Koji Ikegami
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Time Lapse ◽  
Alpha Tubulin ◽  
Endogenous Gene ◽  
Stable Cell Lines ◽  
Long Time ◽  
Endogenous Proteins ◽  
Endogenous Loci ◽  
Time Lapse Imaging

AbstractStable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biological technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5’UTR of the endogenous gene loci. With this 5’UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high knock-in efficiencies ranging from 50 to 80%. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5’UTR is a ‘hotspot’ for targeted insertion of gene sequences and allows the stable expression of tagged proteins from endogenous loci in mammalian cells.

scholarly journals Time-lapse Imaging Reveals Dynamic Relocalization of PP1γ throughout the Mammalian Cell Cycle

2003 ◽  
Vol 14 (1) ◽  
pp. 107-117 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Paul D. Andrews ◽  
Sasala Wickramasinghe ◽  
Judith Sleeman ◽  
Alan Prescott ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Cellular Processes ◽  
Mammalian Cell Cycle ◽  
Hela Cell Lines ◽  
Spatio Temporal ◽  
Time Lapse Imaging

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, α, β/δ, and γ1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1γ or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1γ to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1γ shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1γ also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1γ revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.

scholarly journals Long time-lapse imaging reveals unique features of PARK2/Parkin-mediated mitophagy in mature cortical neurons

Autophagy ◽  
2012 ◽  
Vol 8 (6) ◽  
pp. 976-978 ◽  
Author(s):  
Qian Cai ◽  
Hesham Mostafa Zakaria ◽  
Zu-Hang Sheng
Keyword(s):  
Cortical Neurons ◽  
Time Lapse ◽  
Long Time ◽  
Time Lapse Imaging

scholarly journals Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

Toxics ◽  
2017 ◽  
Vol 5 (3) ◽  
pp. 15 ◽  
Author(s):  
André Jochums ◽  
Elsa Friehs ◽  
Franziska Sambale ◽  
Antonina Lavrentieva ◽  
Detlef Bahnemann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Time Lapse ◽  
Standard Cell ◽  
Nanoparticle Uptake ◽  
Time Lapse Imaging ◽  
Nih 3T3

scholarly journals Composition and Dynamics of Human Mitochondrial Nucleoids

2003 ◽  
Vol 14 (4) ◽  
pp. 1583-1596 ◽  
Author(s):  
Nuria Garrido ◽  
Lorena Griparic ◽  
Eija Jokitalo ◽  
Jorma Wartiovaara ◽  
Alexander M. van der Bliek ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Fission ◽  
Time Lapse ◽  
Mitochondrial Network ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Nucleoids ◽  
Dynamic Structures ◽  
Time Lapse Imaging ◽  
Protein Rich

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied inSaccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.

Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 88-96
Author(s):  
Yu. K. Doronin ◽  
I. V. Senechkin ◽  
L. V. Hilkevich ◽  
M. A. Kurcer
Keyword(s):  
Time Lapse ◽  
Reproductive Technologies ◽  
Human Embryos ◽  
Imaging Data ◽  
Noninvasive Methods ◽  
Topographic Features ◽  
Time Parameters ◽  
Embryo Cleavage ◽  
Time Lapse Imaging ◽  
Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

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