scholarly journals CRISPR-Cas12a–assisted PCR tagging of mammalian genes

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Julia Fueller ◽  
Konrad Herbst ◽  
Matthias Meurer ◽  
Krisztina Gubicza ◽  
Bahtiyar Kurtulmus ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Mammalian Tissue ◽  
Target Enrichment ◽  
Culture Cells ◽  
Endogenous Expression ◽  
Tissue Culture Cells ◽  
Potential Sources ◽  
Linear Dsdna ◽  
Mammalian Genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein–tagged genes.

scholarly journals CRISPR-Cas12a-assisted PCR tagging of mammalian genes

2018 ◽  
Author(s):  
Julia Fueller ◽  
Konrad Herbst ◽  
Matthias Meurer ◽  
Krisztina Gubicza ◽  
Bahtiyar Kurtulmus ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Mammalian Tissue ◽  
Target Enrichment ◽  
Culture Cells ◽  
Endogenous Expression ◽  
Tissue Culture Cells ◽  
Potential Sources ◽  
Linear Dsdna ◽  
Mammalian Genes

AbstractHere we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

1993 ◽  
Vol 51 ◽  
pp. 342-343
Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon
Keyword(s):  
Tissue Culture ◽  
Cell Cycle Progression ◽  
Group Formation ◽  
Sulfhydryl Group ◽  
Mammalian Tissue ◽  
Mitotic Chromosomes ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Transmission Electron

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

scholarly journals Screens Using RNAi and cDNA Expression as Surrogates for Genetics in Mammalian Tissue Culture Cells

2005 ◽  
Vol 70 (0) ◽  
pp. 449-459 ◽  
Author(s):  
J. PEARLBERG ◽  
S. DEGOT ◽  
W. ENDEGE ◽  
J. PARK ◽  
J. DAVIES ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Mammalian Tissue ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Cdna Expression

Cytoplasmic inheritance in mammalian tissue culture cells

In Vitro ◽  
1976 ◽  
Vol 12 (11) ◽  
pp. 758-776 ◽  
Author(s):  
Douglas C. Wallace ◽  
Y. Pollack ◽  
C. L. Bunn ◽  
J. M. Eisenstadt
Keyword(s):  
Tissue Culture ◽  
Cytoplasmic Inheritance ◽  
Mammalian Tissue ◽  
Culture Cells ◽  
Tissue Culture Cells

scholarly journals Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases

2008 ◽  
Vol 183 (4) ◽  
pp. 589-595 ◽  
Author(s):  
Chawon Yun ◽  
Yonggang Wang ◽  
Debaditya Mukhopadhyay ◽  
Peter Backlund ◽  
Nagamalleswari Kolli ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Ribosome Biogenesis ◽  
Mammalian Tissue ◽  
Hematopoietic Malignancies ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Egg Extracts ◽  
Protein B23 ◽  
Sumo Pathway

Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.

scholarly journals Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

2002 ◽  
Vol 13 (12) ◽  
pp. 4401-4413 ◽  
Author(s):  
Ewa Markiewicz ◽  
Thomas Dechat ◽  
Roland Foisner ◽  
Roy. A Quinlan ◽  
Christopher J. Hutchison
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Lamin A ◽  
Dependent Manner ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Green Fluorescent

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathioneS-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.

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