Transgene Site-Specific Integration: Problems and Solutions

2012 ◽  
pp. 3-39 ◽  
Author(s):  
Solenne Bire ◽  
Florence Rouleux-Bonnin
Keyword(s):  
Site Specific ◽  
Site Specific Integration ◽  
Problems And Solutions ◽  
Integration Problems

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

scholarly journals Transcriptional Analysis of the Adeno-Associated Virus Integration Site

2009 ◽  
Vol 83 (23) ◽  
pp. 12512-12525 ◽  
Author(s):  
Nathalie Dutheil ◽  
Els Henckaerts ◽  
Erik Kohlbrenner ◽  
R. Michael Linden
Keyword(s):  
Transcriptional Activity ◽  
Integration Site ◽  
Regulatory Elements ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Complex Interactions ◽  
Site Specific Integration ◽  
Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

Site-specific integration of rotavirus VP6 gene in rabbit β-casein locus by CRISPR/Cas9 system

2019 ◽  
Vol 55 (8) ◽  
pp. 586-597 ◽  
Author(s):  
Hongli Li ◽  
Zhipeng Li ◽  
Ning Xiao ◽  
Xiaoping Su ◽  
Shanshan Zhao ◽  
...  
Keyword(s):  
Site Specific ◽  
Site Specific Integration ◽  
Vp6 Gene ◽  
Rotavirus Vp6

scholarly journals Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

1994 ◽  
Vol 60 (12) ◽  
pp. 4279-4283 ◽  
Author(s):  
M T Alegre ◽  
B Cournoyer ◽  
J M Mesas ◽  
M Guérineau ◽  
P Normand ◽  
...  
Keyword(s):  
Streptomyces Lividans ◽  
Site Specific ◽  
Site Specific Integration

scholarly journals Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus

2020 ◽  
Author(s):  
Bhuvan Pathak ◽  
Vibha Srivastava
Keyword(s):  
Metabolic Engineering ◽  
Rice Genome ◽  
Inducible Promoter ◽  
High Rate ◽  
Site Specific ◽  
Multigene Transformation ◽  
Site Specific Integration ◽  
Targeted Integration ◽  
Transgenic Lines ◽  
Inducible Genes

SummaryEfficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co-integration and co-expression of the genes could present a bottleneck. Recombinase-mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site-specific integration locus is needed. Here, the recombinase technology based on Cre-lox recombination was evaluated for developing multigenic lines harboring constitutively-expressed and inducible genes. Targeted integration of a 5 genes cassette in the rice genome generated a precise full-length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance-stability of the multigene locus. Co-localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other’s expression pattern. In summary, high rate of co-integration and co-expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co-segregating traits.Significance StatementRecombinase-mediated site-specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

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.

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