Comparative fitness assessment of <i>Anopheles stephensi</i> transgenic lines receptive to site‐specific integration

Amenya, Dolphine A.; Bonizzoni, Mariangela; Isaacs, Alison T.; Jasinskiene, Nijole; Chen, Hong; Marinotti, Osvaldo; Yan, Guiyun; James, Anthony A.

doi:10.1111/j.1365-2583.2009.00986.x

Cited by 51 publications

(62 citation statements)

References 17 publications

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“…Technologies used thus far are based on the random integration of transposable elements into the genome; such insertions must be thoroughly characterized with respect to effects on both the fitness of the mosquito (41,42) and the stability/expression of the transgene (43). Although some of this effort can be reduced by reusing integration sites via ΦC31 gene targeting (23,43,44), this still leaves a relatively small portion of the genome currently accessible to manipulation, namely those few regions that have accepted a transposon integration and have been determined to have few undesired effects.…”

Section: Discussionmentioning

confidence: 99%

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Basu

Aryan

Overcash

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

144

129

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Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon-or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

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Section: Discussionmentioning

confidence: 99%

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Basu

Aryan

Overcash

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

144

129

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“…These results support the conclusion that the exogenously derived gypsy insulator-like DNA is functional in this mosquito and contributes to higher and more consistent transgene mRNA abundance. Mosquito embryos from docking lines 44C and 20 containing attP sites for φC31 integrase (18,40) differences were seen in the normalized abundance of DsRed mRNA between each gypsy-containing corresponding pair, although the values in 44C #12 were consistently 9-12% higher than 44C #P2 ( Fig. 4; Tables S3-S5).…”

Section: Resultsmentioning

confidence: 97%

“…stephensi ( Fig. 1) (18,40). The recipient lines were selected because they have single docking-site transgene insertions at different places in the genome and vary visually in the intensity and distribution of their CFP phenotype (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…piggy-Bac often inserts into multiple and distinct sites of the An. stephensi genome during transgenesis experiments (11,18,40). As such, many transgenic lines are actually populations of mosquitoes consisting of individuals with different qualitative and quantitative transgene representations generated by insertion-site effects, linkage, independent assortment, and hemi-or homozygosity.…”

Section: Discussionmentioning

confidence: 99%

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Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi

Carballar-Lejarazú

Jasinskiene

James

2013

Proc. Natl. Acad. Sci. U.S.A.

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Malaria parasites are transmitted to humans by mosquitoes of the genus Anopheles, and these insects are the targets of innovative vector control programs. Proposed approaches include the use of genetic strategies based on transgenic mosquitoes to suppress or modify vector populations. Although substantial advances have been made in engineering resistant mosquito strains, limited efforts have been made in refining mosquito transgene expression, in particular attenuating the effects of insertions sites, which can result in variations in phenotypes and impacts on fitness due to the random integration of transposon constructs. A promising strategy to mitigate position effects is the identification of insulator or boundary DNA elements that could be used to isolate transgenes from the effects of their genomic environment. We applied quantitative approaches that show that exogenous insulator-like DNA derived from the Drosophila melanogaster gypsy retrotransposon can increase and stabilize transgene expression in transposon-mediated random insertions and recombinase-catalyzed, site-specific integrations in the malaria vector mosquito, Anopheles stephensi. These sequences can contribute to precise expression of transgenes in mosquitoes engineered for both basic and applied goals.transgenesis | transposase

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“…stephensi transgenic lines containing phi C31 attP 'docking' sites and expressing ECFP. (Amenya et al 2011).…”

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confidence: 99%

Transgenesis, Paratransgenesis and Transmission Blocking Vaccines to Prevent Insect-Borne Diseases

Ramalho-Ortigão¹,

Coutinho-Abreu²

2012

Integrated Pest Management and Pest Control - Current and Future Tactics

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Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site‐specific integration

Cited by 51 publications

References 17 publications

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi

Transgenesis, Paratransgenesis and Transmission Blocking Vaccines to Prevent Insect-Borne Diseases

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