scholarly journals Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

2014 ◽  
Vol 9 (7) ◽  
pp. 1698-1712 ◽  
Author(s):  
Emilie Pondeville ◽  
Nicolas Puchot ◽  
Janet M Meredith ◽  
Amy Lynd ◽  
Kenneth D Vernick ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Site Specific ◽  
Germline Transformation ◽  
Φc31 Integrase

Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase

2003 ◽  
Vol 21 (3) ◽  
pp. 321-324 ◽  
Author(s):  
Gusztav Belteki ◽  
Marina Gertsenstein ◽  
David W. Ow ◽  
Andras Nagy
Keyword(s):  
Es Cells ◽  
Germline Transmission ◽  
Site Specific ◽  
Mouse Es Cells ◽  
Cassette Exchange ◽  
Φc31 Integrase

Construction of Transgenic Drosophila by Using the Site-Specific Integrase From Phage φC31

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1775-1782
Author(s):  
Amy C Groth ◽  
Matthew Fish ◽  
Roel Nusse ◽  
Michele P Calos
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
P Element ◽  
Drosophila Genome ◽  
S2 Cells ◽  
Precise Integration ◽  
Site Specific ◽  
Attp Site ◽  
Recognition Sites ◽  
Φc31 Integrase

Abstract The φC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. φC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the φC31 integrase system and will likely benefit research in Drosophila and other insects.

scholarly journals Site-specific genetic engineering of the Anopheles gambiae Y chromosome

2014 ◽  
Vol 111 (21) ◽  
pp. 7600-7605 ◽  
Author(s):  
F. Bernardini ◽  
R. Galizi ◽  
M. Menichelli ◽  
P.-A. Papathanos ◽  
V. Dritsou ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Anopheles Gambiae ◽  
Y Chromosome ◽  
Site Specific

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.

scholarly journals Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

BMC Genetics ◽  
2020 ◽  
Vol 21 (S2) ◽  
Author(s):  
Hassan M. M. Ahmed ◽  
Fabienne Heese ◽  
Ernst A. Wimmer
Keyword(s):  
Genetic Engineering ◽  
Control Strategies ◽  
Landing Site ◽  
Drosophila Suzukii ◽  
French Alps ◽  
Germline Transformation ◽  
Φc31 Integrase ◽  
Transgenic Lines ◽  
Vinegar Fly ◽  
Driver Line

Abstract Background The invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing. Results We experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer. Conclusion Our results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

scholarly journals Site-Specific Integration and Expression of an Anti-Malarial Gene in Transgenic Anopheles gambiae Significantly Reduces Plasmodium Infections

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e14587 ◽  
Author(s):  
Janet M. Meredith ◽  
Sanjay Basu ◽  
Derric D. Nimmo ◽  
Isabelle Larget-Thiery ◽  
Emma L. Warr ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Site Specific ◽  
Site Specific Integration

scholarly journals Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae.

1992 ◽  
Vol 12 (11) ◽  
pp. 5102-5110 ◽  
Author(s):  
N J Besansky ◽  
S M Paskewitz ◽  
D M Hamm ◽  
F H Collins
Keyword(s):  
Anopheles Gambiae ◽  
Sequence Similarity ◽  
Mosquito Species ◽  
Target Site ◽  
Rrna Genes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Wild Female ◽  
Site Specific ◽  
Gambiae Complex

Two distinct site-specific retrotransposon families, named RT1 and RT2, from the sibling mosquito species Anopheles gambiae and A. arabiensis, respectively, were previously identified. Both were shown to occupy identical nucleotide positions in the 28S rRNA gene and to be flanked by identical 17-bp target site duplications. Full-length representatives of each have been isolated from a single species, A. gambiae, and the nucleotide sequences have been analyzed. Beyond insertion specificity, RT1 and RT2 share several structural and sequence features which show them to be members of the LINE-like, or non-long-terminal-repeat retrotransposon, class of reverse transcriptase-encoding mobile elements. These features include two long overlapping open reading frames (ORFs), poly(A) tails, the absence of long terminal repeats, and heterogeneous 5' truncation of most copies. The first ORF of both elements, particularly ORF1 of RT1, is glutamine rich and contains long tracts of polyglutamine reminiscent of the opa repeat. Near the carboxy ends, three cysteine-histidine motifs occur in ORF1 and one occurs in ORF2. In addition, each ORF2 contains a region of sequence similarity to reverse transcriptases and integrases. Alignments of the protein sequences from RT1 and RT2 reveal 36% identity over the length of ORF1 and 60% identity over the length of ORF2, but the elements cannot be aligned in the 5' and 3' noncoding regions. Unlike that of RT2, the 5' noncoding region of RT1 contains 3.5 copies of a 500-bp subrepeat, followed by a poly(T) tract and two imperfect 55-bp subrepeats, the second spanning the beginning of ORF1. The pattern of distribution of these elements among five siblings species in the A. gambiae complex is nonuniform. RT1 is present in laboratory and wild A. gambiae, A. arabiensis, and A. melas but has not been detected in A. quadriannulatus or A. merus. RT2 has been detected in all available members of the A. gambiae complex except A. merus. Copy number fluctuates, even among the offspring of individual wild female A. gambiae mosquitoes. These findings reflect a complex evolutionary history balancing gain and loss of copies against the coexistence of two elements competing for a conserved target site in the same species for perhaps millions of years.

Sign in / Sign up

Export Citation Format

Share Document