Generation of transgenic fly stocks

Transgenic flies are usually obtained from the microinjection of a P-element based transformation vector together with a helper plasmid into preblastoderm stage embryos. The P-element is a natural transposon ocurring in Drosophila. It features 31 bp inverted terminal repeats and 11 bp inverted subterminal repeats as the major cis-acting elements required for tansposition, which flank the transposase encoding gene that consists of four exons (Castro and Carareto 2004). As not more than 138 bp at the 5’ end and 216 bp at the 3’ end of the P-element are required for transposition in cis (Beall and Rio 1997), the transposase gene can be removed from the P-element. Replacing it by a selectable marker, usually a w+ allele confering red eyes to w mutant flies that otherwise have white eyes, and an MCS to take up exogenous DNA yields a transformation vector that is able to transpose from the injected plasmid into the genome. Moreover, removing the transposase gene from the P-element prevents it from continued transpositions and allows for stable transgenic stocks. For the initial transposition from the injected plasmid into the genome, the transposase is supplied from a co-injected helper plasmid. Transformation vector and helper plasmid are injected into the posterior pole of the embryo before cellularization. The Drosophila embryo develops as a syncytium for the first 1.5 h after fertilization (Allis et al. 1977). Upon cellularization, the nuclei deposited at the posterior pole of the syncytium are incorporated into the pole cells, which are the precursors of the germ cells. A transgene inserted into the genome of a pole cell will therefore be passed on to gametes generated by the fly arising from the injected embryo and eventually to its progeny, which will then consist entirely of transgenic cells. These transgenic animals can be identified by means of the selectable marker included in the transformation vector.

Microinjections

In order to generate transgenic flies with gogo constructs, embryos homozygous for the w1118 mutation, which causes white eye color, were collected for 30’ at 25 °C on apple juice agar plates from a population cage. To dechorionate the embryos, they were treated with 50 % household bleach for 2’ directly on the apple juice agar plates, then poured onto a vacuum-driven membrane filter and rinsed with plenty of tap water. The dechorionated embryos were manually lined up on the filter side by side using a fine brush. Typically, 200 embryos were used per construct. The embryos were transferred onto a coverslip that had been moistened with Scotch glue in heptane and air-dried before. Scotch glue in heptane was obtained by simply leaving Scotch Tape in n-heptane for some days with some agitation. The coverslip with the embryos was placed onto a microscope slide and the embryos were dried in a large Petri dish over silica gel for approximately

34 MATERIALS AND METHODS

15’. Then, the embryos were covered with halocarbon oil, and a mixture of 0.6 µg/µl of the plasmid carrying the particular transgenic construct and 0.2 µg/µl of the helper plasmid in water was injected using a FemtoJet microinjector from Eppendorf (Hamburg, Germany). After injection, the coverslip with the embryos was transfered to a vial with fresh fly food.

Microinjections of plasmids with hts constructs into w1118 embryos were performed by BestGene (Chino Hills, California).

Balancing

The flies developing from the injected embryos were individually crossed to w1118 flies. Successful germline transformation resulted in red-eyed flies in the F1 generation. To breed stable transgenic stocks and to determine at which chromosome the transformation vector was inserted, red-eyed flies were crossed to flies from balancer stocks. If possible, the balancer chromosomes were removed from the transgenic stocks to obtain homozygous flies.

The following fly stocks were used to generate transgenic fly stocks:

w1118 Suzuki Lab (M45)

w1118 / Y, hs-hid Suzuki Lab (M46)

y* w* / Y, hs-hid ; Pin* / CyO this work, S011

y* w* / Y, hs-hid ; sensLy-1 / TM3, Sb1 this work, S012

List of transgenic fly stocks that were generated

All transgenic fly stocks that were generated and used in this work are listed below.

genotype plasmid chromosome promoter protein

w*, P{GMR-gogo-Myc}XA pGMR-gogo-Myc X GMR Gogo-Myc

y* w* ; P{GMR-gogo-Myc}2A pGMR-gogo-Myc 2 GMR Gogo-Myc

w*, P{GMR-gogo C-Myc}XA pGMR-gogo C-Myc X GMR Gogo C-Myc

y* w* ; P{GMR-gogo C-Myc}2A pGMR-gogo C-Myc 2 GMR Gogo C-Myc

y* w* ; P{GMR-gogocyto-Myc}3B pGMR-gogocyto-Myc 3 GMR Gogocyto-Myc

w*, P{GMR-Add1}XA pGMR-Add1 X GMR Add1

y* w* ; P{GMR-Add1}2A pGMR-Add1 2 GMR Add1

y* w* ; P{GMR-Add1}3A pGMR-Add1 3 GMR Add1

y* w* ; P{GMR-Add1}3B pGMR-Add1 3 GMR Add1

w*, P{GMR-ShAdd}XA pGMR-ShAdd X GMR ShAdd

y* w* ; P{GMR-ShAdd}3A pGMR-ShAdd 3 GMR ShAdd

y* w* ; P{GMR-ShAdd}3B pGMR-ShAdd 3 GMR ShAdd

genotype plasmid chromosome promoter protein

y* w* ; P{GMR-htsPD}3A pGMR-htsPD 3 GMR HtsPD

y* w* ; P{GMR-htsPD}3B pGMR-htsPD 3 GMR HtsPD

y* w* ; P{UAS-gogo-Myc}2B / CyO pUAST-gogo-Myc 2 UAS Gogo-Myc

y* w* ; P{UAS-gogo-Myc}3B pUAST-gogo-Myc 3 UAS Gogo-Myc

y* w* ; P{UAS-Add1-Myc}2A / CyO pUAST-Add1-Myc 2 UAS Add1-Myc

y* w* ; P{UAS-Add1-Myc}3A pUAST-Add1-Myc 3 UAS Add1-Myc

y* w* ; P{UAS-ShAdd-His}2A pUAST-ShAdd-His 2 UAS ShAdd-His

y* w* ; P{UAS-ShAdd-His}2B / CyO pUAST-ShAdd-His 2 UAS ShAdd-His

y* w* ; P{UAS-hts472-His}3A pUAST-hts472-His 3 UAS Hts472-His

y* w* ; P{UAS-hts472-His}3B pUAST-hts472-His 3 UAS Hts472-His Table 2.1: List of transgenic fly stocks that were generated

The genotype of each stock is denoted in the first column. The second column states the plasmid that was injected and the third column the chromosome at which the transformation vector was inserted. The fourth column quotes the promoter contained in the transgenic construct to control the expression of the protein listed in the fifth column.