Submission Number: KOS-4-11-7

Abstract Number: 305

TRANSGENIC HOUSE FLY Musca domestica WITH NEOMYCIN RESISTANCE.

VS Kosorukov, TA Antipova, MI Prokofiev and LK Ernst

Research and Production Biotechnology Center for Animal Husbandry, Russian Academy of Agricultural Science, Gorki Leninskie, Moscow, Russia ¹

Abstract:
The transformation of different insect species became possible after discovery of hobo-like mobile elements. There are many applications for transgenic insects, for example, manipulations with natural populations and genetic researches. Musca domestica is a very interesting object for this applications. Two gene vectors were constructed: A) pHerm[NeoR] with inverted terminal repeats of Hermes transposable element and gene of neomycin resistance NeoR under control of Drosophila melanogaster hsp-70-promotor; B) pHerm[NeoR,GFP] in addition carry EGFP gene under Drosophila pseudoobscura hsp-82 promoter. About 100 pl of gene vector in mixture with helper with nonmobile transposase (0.2 mkg/ml and 0.05 mkg/ml respectively) were microinjected in preblastoderma embryos of Musca domestica using adapted technique. The first generation was reared on usual medium. Flies received were crossed with wild one and embryos were placed on agar medium with 500 mg/l G418. Survived larvae start separate lines. Survived lines were tested for transgene by PCR. 15262 embryo were microinjected with A and 186 imago (1.22%) were received. 16 (8.6%) of them gave progeny on medium with G418. PCR confirmed the fact of vector integration in all survived lines. Inheritance of transgene was retraced in 10 generations. 1365 embryo were microinjected with B and from 22 imago (1.6%) 3 transgenic lines were started. High self luminescence of Musca d. was the course that perfect visual detection of GFP was possible only in one line. Starting from third generation nontransgenic larvae in nongomozygote lines began to survive on selective medium in concentrations up to 1000 mg/l of because of habituation of wild larvae to G418. Concentrations more than 1000 mg/l G418 are lethal for both wild and transgenic larvae whose selection failed due to low level of hsp-70 promoter expression in Musca d. This results showed that NeoR and GFP may be used as markers for transgene selection in Musca d., but stronger promoters were needed. .

Keywords: Musca domestica, transgene, insects, mobile element, neomycin resistanse, GFP.

This abstract is being presented at: 8:00 AM in session:
Gene Regulation and Function I