scholarly journals Agrobacterium tumefaciens-Mediated Transformation of NHEJ Mutant Aspergillus nidulans Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers

2021 ◽  
Vol 7 (11) ◽  
pp. 961
Author(s):  
Virginia Casado-del Castillo ◽  
Andrew P. MacCabe ◽  
Margarita Orejas
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Aspergillus Nidulans ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Attractive Alternative ◽  
Nutritional Requirement ◽  
Position Effects ◽  
Site Specific ◽  
Enzyme Preparations ◽  
Site Specific Integration

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants.

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.

Transgene Coplacement and High Efficiency Site-Specific Recombination With the Cre/loxP System in Drosophila

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 715-726 ◽  
Author(s):  
Mark L Siegal ◽  
Daniel L Hartl
Keyword(s):  
Germ Cells ◽  
Maternal Effect ◽  
High Efficiency ◽  
Loxp Site ◽  
Position Effects ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Bacteriophage P1 ◽  
Eye Color ◽  
F2 Generation

Abstract Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mosl promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F1 progeny. Excision in germ cells of the F1 yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F2 generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.

scholarly journals Site-Specific Integration of Agrobacterium tumefaciens T-DNA via Double-Stranded Intermediates

2003 ◽  
Vol 133 (3) ◽  
pp. 1011-1023 ◽  
Author(s):  
Tzvi Tzfira ◽  
Leah Renée Frankman ◽  
Manjusha Vaidya ◽  
Vitaly Citovsky
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Site Specific ◽  
Site Specific Integration

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.

A Characteristic-Featured Shock Wave Indicator for Simulating High-Speed Inviscid Flows on 3D Unstructured Mesh

2021 ◽  
Author(s):  
Yiwei Feng ◽  
Tiegang Liu ◽  
Xiaofeng He ◽  
Bin Zhang ◽  
Kun Wang
Keyword(s):  
Shock Wave ◽  
High Speed ◽  
Unstructured Mesh ◽  
High Efficiency ◽  
Flow Simulation ◽  
Attractive Alternative ◽  
Inviscid Flows ◽  
Speed Flow ◽  
Flow Compression ◽  
Shock Processing

Abstract In this work, we extend the characteristic-featured shock wave indicator based on artificial neuron training to 3D high-speed flow simulation on unstructured mesh. The extension is achieved through dimension splitting. This leads to that the proposed indicator is capable of identifying regions of flow compression in any direction. With this capability, the indicator is further developed to combine with h-adaptivity of mesh refinement to improve resolution with less computational costs. The present indicator provided an attractive alternative for constructing high-resolution, high-efficiency shock-processing method to simulate high-speed inviscid flows.

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
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