scholarly journals 194. Characteristics of Site-Specific Integration as Mediated by PhiC31 Integrase in the Human Genome

2005 ◽  
Vol 11 ◽  
pp. S76
Keyword(s):  
Human Genome ◽  
Phic31 Integrase ◽  
Site Specific ◽  
Site Specific Integration

scholarly journals Site-Specific Integration of Functional Transgenes into the Human Genome by Adeno/AAV Hybrid Vectors

2004 ◽  
Vol 10 (4) ◽  
pp. 660-670 ◽  
Author(s):  
Alessandra Recchia ◽  
Laura Perani ◽  
Daniela Sartori ◽  
Clelia Olgiati ◽  
Fulvio Mavilio
Keyword(s):  
Human Genome ◽  
Site Specific ◽  
Hybrid Vectors ◽  
Site Specific Integration

scholarly journals The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Coral González-Prieto ◽  
Richard Gabriel ◽  
Christoph Dehio ◽  
Manfred Schmidt ◽  
Matxalen Llosa
Keyword(s):  
Human Genome ◽  
Fold Increase ◽  
Human Cells ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Dna Complexes ◽  
Site Specific ◽  
Type Iv ◽  
Site Specific Integration ◽  
A Site

ABSTRACT Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

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.

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

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

Genetics ◽  
2021 ◽  
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

Abstract We 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 two-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 Erratum: Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

2006 ◽  
Vol 3 (9) ◽  
pp. 763-763
Author(s):  
Louis J Nkrumah ◽  
Rebecca A Muhle ◽  
Pedro A Moura ◽  
Pallavi Ghosh ◽  
Graham F Hatfull ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Site Specific ◽  
Site Specific Integration
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