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Publication Type: Proceedings Publication Acceptance Date: 7/15/2004 Publication Date: 9/20/2004 Citation: Allen, M.L. 2004. Mass rearing transgenic insects: The importance of screening. Internet Web Page. Available: http://pewagbiotech.org/events/0920/ Interpretive Summary: Technical Abstract: Genetically modified insects may be utilized in future release programs. Successful insect transformation requires appropriate molecular tools and appropriate insect handling skills. Establishment of transgenic strains requires vigilant insect colony tending. Beyond this, any successful release program will depend upon the ability to mass rear healthy transgenic laboratory colonies. These colonies will require standardized maintenance procedures and specialized assays to validate each insect application, and to select the most appropriate strain(s). For every insect species, procedures and assays will be unique, and new challenges will emerge. Some species and even strains of insects are inherently more amenable to laboratory rearing than others. For two very different species of insect, the Southern house mosquito Culex quinquefasciatus and the New World screwworm Cochliomyia hominivorax, successful transformation protocols and successful strain establishment were accomplished. In both cases, isolation of distinct fluorescence expression phenotypes at a developmental stage that allowed insect handling for screening purposes facilitated strain establishment. Screening was the process of removing non-transgenic insects from the colony and sorting to isolate distinct phenotypes. The Southern house mosquito is a vector for lymphatic filariasis, West Nile virus, and other filarial and viral diseases. Two successful transformation efforts produced three distinct transgenic strains. Transgenic strains incorporating the actin5cEGFP transgene could be screened at any developmental stage because fluorescence was present at all stages, and the insect cuticle is not opaque. The transgenic strain incorporating the actin88FGFP transgene could only be screened at the pupa stage. Earlier developmental stages were not fluorescent. Adult mosquitoes were never screened because of flight/escape risk. The New World screwworm is a pest of livestock and other warm-blooded creatures (including humans). It has been successfully eradicated from the entire North American continent. The eradication program was the first implementation of the sterile insect technique (SIT). This insect is currently prevented from reinfesting North America by the ongoing release of sterile, irradiated adult flies at the barrier zone in Panama. Using a piggyBac TE system, a pUbEGFP transgene was introduced to the germ line of a laboratory strain (P95) of screwworms, resulting in eight transgenic strains with distinct visible phenotypes. The strains could only be screened prior to pupation because the pupa and imago cuticle was opaque. Just prior to formation of the pupal cuticle, the larva exits the rearing media (as a 'crawler'). This was established as the screening stage. These two examples represent insect species that were relatively easy to establish as laboratory culture, but required intensive maintenance. Nevertheless, it was possible to integrate the transgenic screening process into the rearing schedule. Expression of the marker during a stage or point of development at which the insects could be easily sorted was crucial. |
