Dataset Information

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

ABSTRACT: Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.

SUBMITTER: Meza JS

PROVIDER: S-EPMC4193812 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Meza José S JS Díaz-Fleischer Francisco F Sánchez-Velásquez Lázaro R LR Zepeda-Cisneros Cristina Silvia CS Handler Alfred M AM Schetelig Marc F MF

PloS one 20141010 10

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in s ...[more]

PMID: 25303238

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Project description:Reliable marking systems are critical to the prospective field release of transgenic insect strains. This is to unambiguously distinguish released insects from wild insects in the field that are collected in field traps, and tissue-specific markers, such as those that are sperm-specific, have particular uses such as identifying wild females that have mated with released males. For tephritid fruit flies such as the Mexican fruit fly, Anastrepha ludens, polyubiquitin-regulated fluorescent protein body markers allow transgenic fly identification, and fluorescent protein genes regulated by the spermatocyte-specific ?2-tubulin promoter effectively mark sperm. For sterile male release programs, both marking systems can be made male-specific by linkage to the Y chromosome.An A. ludens wild type strain was genetically transformed with a piggyBac vector, pBXL{PUbnlsEGFP, As?2tub-DsRed.T3}, having the polyubiquitin-regulated EGFP body marker, and the ?2-tubulin-regulated DsRed.T3 sperm-specific marker. Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (YEGFP strain) expressed only PUbnlsEGFP. This insertion was remobilized by transposase helper injection, which resulted in three new autosomal insertion lines that expressed both markers. This indicated that the original Y-linked As?2tub-DsRed.T3 marker was functional, but specifically suppressed on the Y chromosome. The PUbnlsEGFP marker remained effective however, and the YEGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp+) gene onto the Y, which was then introduced into the bp- mutant strain. This allows the mechanical separation of mutant female black pupae from male brown pupae, that can be identified as adults by EGFP fluorescence.A Y-linked insertion of the pBXL{PUbnlsEGFP, As?2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(YEGFP/bp+), allowing the identification of male adults when used in sterile male release programs for population control. A unique observation was that expression of the As?2tub-DsRed.T3 sperm-specific marker, which was functional in autosomal insertions, was specifically suppressed in the Y-linked insertion. This may relate to the Y chromosomal regulation of male-specific germ-line genes in Drosophila.

Dataset Information

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Publications

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

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