A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

1995 ◽  
Vol 27 (6) ◽  
pp. 536-540 ◽  
Author(s):  
R. J. Gouka ◽  
J. G. M. Hessing ◽  
H. Stam ◽  
W. Musters ◽  
C. A. M. J. J. van den Hondel
Keyword(s):  
Aspergillus Awamori ◽  
Integration System ◽  
Site Specific ◽  
Site Specific Integration ◽  
Novel Strategy ◽  
A Site

scholarly journals Efficient Integration of Recombinant Adeno-Associated Virus DNA Vectors Requires a p5-rep Sequence in cis

2002 ◽  
Vol 76 (11) ◽  
pp. 5411-5421 ◽  
Author(s):  
Nicola J. Philpott ◽  
Catherine Giraud-Wali ◽  
Carolyn Dupuis ◽  
Janette Gomos ◽  
Henry Hamilton ◽  
...  
Keyword(s):  
First Generation ◽  
Promoter Sequence ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Enhancer Element ◽  
Site Specific ◽  
Site Specific Integration ◽  
Gene Therapy Vectors ◽  
A Site

ABSTRACT The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

scholarly journals Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1

2002 ◽  
Vol 184 (7) ◽  
pp. 1859-1864 ◽  
Author(s):  
Hee-Youn Yang ◽  
Young-Woo Kim ◽  
Hyo-Ihl Chang
Keyword(s):  
Pcr Amplification ◽  
Temperate Phage ◽  
Vector System ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Integration Vector ◽  
Site Specific Integration ◽  
A Site

ABSTRACT The genome of temperate phage φFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage φFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 103 transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering in E. faecalis and in other hosts.

scholarly journals A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus

2019 ◽  
Vol 10 (2) ◽  
pp. 605-611
Author(s):  
Fang Tao ◽  
Kai Zhao ◽  
Qianqian Zhao ◽  
Fangzhi Xiang ◽  
Guomin Han
Keyword(s):  
Aspergillus Flavus ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Fungal Growth ◽  
Egfp Expression ◽  
Regulation Mechanism ◽  
Site Specific ◽  
Site Specific Integration ◽  
Foreign Dna ◽  
Novel Strategy

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2-lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2-locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus. To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2-locus in the genome of Aspergillus.

A new site-specific integration system for mycobacteria

Tuberculosis ◽  
2005 ◽  
Vol 85 (5-6) ◽  
pp. 317-323 ◽  
Author(s):  
Jeffrey Murry ◽  
Christopher M. Sassetti ◽  
Jonathan Moreira ◽  
James Lane ◽  
Eric J. Rubin
Keyword(s):  
Integration System ◽  
Site Specific ◽  
Site Specific Integration

scholarly journals The Site-Specific Integration System of the TemperateStreptococcus thermophilusBacteriophage φSfi21

Virology ◽  
1997 ◽  
Vol 237 (1) ◽  
pp. 148-158 ◽  
Author(s):  
Anne Bruttin ◽  
Sophie Foley ◽  
Harald Brüssow
Keyword(s):  
Integration System ◽  
Site Specific ◽  
Site Specific Integration

scholarly journals Vectors for efficient and high-throughput construction of fluorescent drosophila reporters using the PhiC31 site-specific integration system

genesis ◽  
2010 ◽  
Vol 48 (7) ◽  
pp. 452-456 ◽  
Author(s):  
Aurelia L. Boy ◽  
Zongzhao Zhai ◽  
Anette Habring-Müller ◽  
Yvonne Kussler-Schneider ◽  
Petra Kaspar ◽  
...  
Keyword(s):  
High Throughput ◽  
Integration System ◽  
Site Specific ◽  
Site Specific Integration

scholarly journals Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

Circulation ◽  
2012 ◽  
Vol 126 (11_suppl_1) ◽  
pp. S20-S28 ◽  
Author(s):  
F. Lan ◽  
J. Liu ◽  
K. H. Narsinh ◽  
S. Hu ◽  
L. Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genetic Modification ◽  
Cardiac Stem Cells ◽  
Integration Technique ◽  
Site Specific ◽  
Site Specific Integration ◽  
A Site

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 Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage φC31 Integrase

2001 ◽  
Vol 21 (12) ◽  
pp. 3926-3934 ◽  
Author(s):  
Bhaskar Thyagarajan ◽  
Eric C. Olivares ◽  
Roger P. Hollis ◽  
Daniel S. Ginsburg ◽  
Michele P. Calos
Keyword(s):  
Mammalian Cells ◽  
Chromosome Engineering ◽  
Site Specific ◽  
Site Specific Integration ◽  
Attachment Sites ◽  
Phage Integrase ◽  
Φc31 Integrase ◽  
Efficient Integration ◽  
A Site ◽  
Human And Mouse

ABSTRACT We previously established that the phage φC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phageattP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native “pseudo”attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

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