scholarly journals Establishing isogenic inducible cell lines using founder reporter lines and recombinase-mediated cassette exchange

BioTechniques ◽  
2013 ◽  
Vol 55 (5) ◽  
Author(s):  
Bin Guan ◽  
Tae Magomi ◽  
Tian-Li Wang ◽  
Ie-Ming Shih
Keyword(s):  
Cell Lines ◽  
Cassette Exchange

Utilization of recombinase mediated cassette exchange (RMCE) for the generation of recombinant CHO cell lines with defined expression properties

2014 ◽  
Vol 31 ◽  
pp. S4
Author(s):  
Martina Baumann ◽  
Elisabeth Gludovacz ◽  
Sabine Vcelar ◽  
Nicole Borth
Keyword(s):  
Cell Lines ◽  
Cho Cell ◽  
Cassette Exchange

scholarly journals Generation of Drosophila attP containing cell lines using CRISPR-Cas9

2021 ◽  
Author(s):  
Daniel Mariyappa ◽  
Arthur Luhur ◽  
Danielle Overton ◽  
Andrew C Zelhof
Keyword(s):  
Cell Lines ◽  
Single Copy ◽  
Drosophila Genome ◽  
Phic31 Integrase ◽  
Drosophila Cell ◽  
In Vivo Experiments ◽  
Cassette Exchange ◽  
Transgenic Cell ◽  
Embryonic Origin

Abstract The generation of Drosophila stable cell lines have become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here we describe a toolkit and procedure that combines CRISPR/Cas9 and the PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.

scholarly journals Generation of Drosophila attP containing cell lines using CRISPR-Cas9

2021 ◽  
Author(s):  
Andrew C. Zelhof ◽  
Daniel Mariyappa ◽  
Arthur Luhur ◽  
Danielle Overton
Keyword(s):  
Cell Lines ◽  
Single Copy ◽  
Minimal Change ◽  
Genetic Screens ◽  
Phic31 Integrase ◽  
Cassette Exchange ◽  
Transgenic Cell ◽  
Cell Clones ◽  
Embryonic Origin

The generation of Drosophila stable cell lines have become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here we describe a toolkit and procedure that combines CRISPR/Cas9 and the PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.

scholarly journals Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

2010 ◽  
Vol 10 (1) ◽  
pp. 92 ◽  
Author(s):  
Susan J Morris ◽  
Daniel C Farley ◽  
Keith N Leppard
Keyword(s):  
Cell Lines ◽  
Cassette Exchange ◽  
Adenovirus Vectors

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: Predictability and efficiency by transgene exchange

2006 ◽  
Vol 124 (2) ◽  
pp. 457-468 ◽  
Author(s):  
A.S. Coroadinha ◽  
R. Schucht ◽  
L. Gama-Norton ◽  
D. Wirth ◽  
H. Hauser ◽  
...  
Keyword(s):  
Cell Lines ◽  
Retroviral Vector ◽  
Cassette Exchange ◽  
Producer Cell

scholarly journals Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghuvir Viswanatha ◽  
Enzo Mameli ◽  
Jonathan Rodiger ◽  
Pierre Merckaert ◽  
Fabiana Feitosa-Suntheimer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Mosquito Species ◽  
Mosquito Cell ◽  
Exchange System ◽  
Public Health Burden ◽  
Cassette Exchange ◽  
Mosquito Cells ◽  
Mosquito Cell Lines ◽  
Genome Scale

AbstractMosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.

scholarly journals Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

2021 ◽  
Author(s):  
Raghuvir Viswanatha ◽  
Enzo Mameli ◽  
Jonathan Rodiger ◽  
Pierre Merckaert ◽  
Fabiana Feitosa-Suntheimer ◽  
...  
Keyword(s):  
Public Health ◽  
Cell Lines ◽  
Mosquito Species ◽  
Screening Tools ◽  
Exchange System ◽  
Public Health Burden ◽  
Cassette Exchange ◽  
Health Burden ◽  
Mosquito Cell Lines ◽  
Genome Scale

Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.

scholarly journals Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0161471 ◽  
Author(s):  
Morten M. Callesen ◽  
Martin F. Berthelsen ◽  
Sira Lund ◽  
Annette C. Füchtbauer ◽  
Ernst-Martin Füchtbauer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Reporter Cell ◽  
Cassette Exchange

scholarly journals Targeted insertion in well-characterized Drosophila cell lines using φC31 integrase

2015 ◽  
Author(s):  
Lucy Cherbas ◽  
Jennifer F. Hackney ◽  
Lei Gong ◽  
Claire Salzer ◽  
Eric Mauser ◽  
...  
Keyword(s):  
Cell Lines ◽  
P Element ◽  
Drosophila Cell ◽  
Cassette Exchange ◽  
Basal Expression ◽  
Φc31 Integrase ◽  
Clonal Lines ◽  
Insulator Elements ◽  
Transformation Methods ◽  
P Element Transformation

We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with and without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrated the technology by integrating a cassette containing a Cu++-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays ??? a major emphasis of cell-based studies.

(EB) Virus: Latency and Expression in Transplanted and Cultured Human Cells

1971 ◽  
Vol 29 ◽  
pp. 368-369
Author(s):  
B. G. Uzman ◽  
M. M. Kasac ◽  
H. Saito ◽  
A. Krishan
Keyword(s):  
Cell Lines ◽  
Primary Cultures ◽  
Virus Particles ◽  
Barr Virus ◽  
Virus Latency ◽  
Virus Surveillance ◽  
Cultured Human Cells ◽  
Epstein Barr ◽  
Serial Transplantation ◽  
Tubular Arrays

In conjunction with the cultivation and transplantation of cells from human tumors by the Programs of Microbiology and Immunogenetics, virus surveillance by electron microscopy has been routinely employed. Of particular interest in this regard have been 3 cell lines cultured from lymph nodes or spleen of 2 patients with Hodgkin's disease and 1 patient with Letterer-Siwe's disease. Each of these cell lines when transplanted in Syrian hamster neonates conditioned with anti-lymphocyte serum grew as serially transplantable tumors; from such transplants of the 3 cell lines cell cultures were retrieved.Herpes type virus particles (Figs. 1, 2, 3) were found in the primary cultures of all three lines, in frozen thawed aliquots of same, and in cultures retrieved from their tumors growing by serial transplantation in hamsters. No virus was detected in sections of 25 of the serially transplanted tumors. However, in 10 such tumors there were repeated instances of tubular arrays in the cisternae of the endoplasmic reticulum (Fig. 4). On serologic examination the herpes virus was shown to be the Epstein-Barr virus.

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