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

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.

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Generation of Drosophila attP containing cell lines using CRISPR-Cas9

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab161 ◽

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.

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

BioTechniques ◽

10.2144/000114098 ◽

2013 ◽

Vol 55 (5) ◽

Cited By ~ 1

Author(s):

Bin Guan ◽

Tae Magomi ◽

Tian-Li Wang ◽

Ie-Ming Shih

Keyword(s):

Cell Lines ◽

Cassette Exchange

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Transformation of Drosophila Cell Lines

Baculovirus and Insect Cell Expression Protocols - Methods in Molecular Biology™ ◽

10.1007/978-1-59745-457-5_16 ◽

2007 ◽

pp. 317-340 ◽

Cited By ~ 7

Author(s):

Lucy Cherbas ◽

Peter Cherbas

Keyword(s):

Cell Lines ◽

Drosophila Cell

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Analysis of transcriptional regulation of the s38 chorion gene of Drosophila by P element-mediated transformation

Development ◽

10.1242/dev.83.supplement.137 ◽

1984 ◽

Vol 83 (Supplement) ◽

pp. 137-146

Author(s):

Laura Kalfayan ◽

Barbara Wakimoto ◽

Allan Spradling

Keyword(s):

Transcriptional Regulation ◽

P Element ◽

Developmental Expression ◽

Developmental Profile ◽

E Coli ◽

Flanking Sequences ◽

Dna Fragment ◽

P Element Transformation ◽

Lac Z

Transcriptional regulation of the s38 chorion gene was studied using P element-mediated germline transformation. A 5·27 kb DNA fragment containing the s38 gene and 5′- and 3′-flanking sequences, was tested for its ability to be transcribed with correct developmental specificity. Five single-insert transformed lines were generated by microinjection of this DNA fragment cloned into a marked P element transformation vector. In each line, the transformed gene was transcribed according to the precise developmental pattern followed by the native s38 gene. The 1·3 kb at the 5′ end of this tested fragment was fused to the E. coli lac z gene. This fragment was also capable of initiating transcription of E. coli lac z RNA with the developmental profile of the native s38 gene. In vitro deletion studies are underway to determine which sequences in the 1·3 kb fragment are necessary for regulating the developmental expression of the gene.

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