In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange

2013 ◽  
Vol 43 (11) ◽  
pp. 997-1008 ◽  
Author(s):  
Dingpei Long ◽  
Aichun Zhao ◽  
Longxia Xu ◽  
Weijian Lu ◽  
Qing Guo ◽  
...  
Keyword(s):  
Phic31 Integrase ◽  
Site Specific ◽  
Cassette Exchange ◽  
Site Specific Integration

scholarly journals Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

2009 ◽  
Vol 151 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
Zhongsen Li ◽  
Aiqiu Xing ◽  
Bryan P. Moon ◽  
Richard P. McCardell ◽  
Kelly Mills ◽  
...  
Keyword(s):  
Site Specific ◽  
Cassette Exchange ◽  
Site Specific Integration

scholarly journals Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration

2007 ◽  
Vol 82 (5) ◽  
pp. 2590-2593 ◽  
Author(s):  
Mickaël Guilbaud ◽  
Gilliane Chadeuf ◽  
Fabio Avolio ◽  
Achille François ◽  
Philippe Moullier ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Virus Type ◽  
Promoter Region ◽  
Raav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Human Chromosome 19

ABSTRACT The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.

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 Scarless engineering of the Drosophila genome near any site-specific integration site

2020 ◽  
Author(s):  
Siqian Feng ◽  
Shan Lu ◽  
Wesley B. Grueber ◽  
Richard S. Mann
Keyword(s):  
Integration Site ◽  
Landing Site ◽  
Genomic Sequences ◽  
Efficient Technique ◽  
Drosophila Genome ◽  
Step Method ◽  
Phic31 Integrase ◽  
Site Specific ◽  
Site Specific Integration ◽  
Landing Sites

AbstractWe describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This 2-step method combines phiC31 integrase mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

scholarly journals Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

2000 ◽  
Vol 74 (19) ◽  
pp. 8831-8842 ◽  
Author(s):  
Stefania Lamartina ◽  
Gennaro Ciliberto ◽  
Carlo Toniatti
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
A Site ◽  
First Time ◽  
Sequence Constraints

ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS andtrs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

2013 ◽  
Vol 48 (3) ◽  
pp. 265-273 ◽  
Author(s):  
Naoyuki Yonemura ◽  
Toshiki Tamura ◽  
Keiro Uchino ◽  
Isao Kobayashi ◽  
Ken-ichiro Tatematsu ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Phic31 Integrase ◽  
Site Specific ◽  
Site Specific Integration ◽  
Silkworm Bombyx Mori
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