A new strategy for efficient in vivo screening of mutagenized Drosophila embryos

Abstract

The analysis of mutants is an indispensable approach towards characterizing gene function. Combining several tools of Drosophila genetics, we designed a new strategy for a mutagenesis screen which is fast, easy-toapply, and cheap. The combination of a cell-specific Gal4 line with an upstream activating sequence-green fluorescent protein (UAS-GFP) allows the in vivo detection of the cells or tissues of interest without the need for fixation and staining. To further simplify and accelerate the screening procedure, we generated recombinant flies that carry the Gal80 transgene in balancer chromosomes. Gal80 inactivates Gal4; and thus prevents GFP-expression during embryonic and postembryonic development in all individuals carrying the balancer chromosomes. This allows for an easy distinction in vivo between heterozygous and homozygous mutants, the latter being the only ones expressing GFP. Since most of the fly strains and balancer chromosomes can be substituted, this method is suitable for nearly any mutagenesis screen that does not have major restrictions.

Refinement of Tools for Targeted Gene Expression in Drosophila

“…The second limitation is the stoichiometric requirement for GAL80. In cases where GAL80 is driven from a strong promoter, such as a-1 tubulin, a single transgene is sufficient to suppress GAL4-mediated transcription (Lee and Luo 1999;Vef et al 2006); however, we found that when GAL4 and GAL80 are driven under the same enhancer, a single copy of GAL80 may be insufficient, especially when the vector utilizes the same, mRNA-destabilizing hsp70 39-UTR as used in the GAL4 constructs. This problem can be fixed by using two copies of GAL80, either by generating a stock bearing GAL80 in two locations or by building a tandem construct with two copies of GAL80 in one insertion.…”

GBF1 (Gartenzwerg)-dependent secretion is required for Drosophila tubulogenesis

“…The EMS allele S4-50 was generated in the lab of C. Klämbt (Hummel et al, 1999) and identified by us in a genetic screen for heart mutants (Albrecht et al, 2006). The EMS-induced mutant garz EMS667 was generated in the laboratory of G. Technau and identified in a screen for nervous system mutants and verified as a garz allele in this study and by O. Vef and B. Altenhein (Vef et al, 2006). The garz EP(2)2028 allele was generated in P. Rørth's lab and obtained from the Szeged Stock Center (Rørth, 1996).…”

“…, was isolated in the Technau laboratory (Vef et al, 2006). Transheterozygous animals with the genotype S4-50/EMS667, S4-50/EP(2)2028 or any other mutant combination are 100% lethal and show the same phenotypes as described in the following sections.…”

Mitoferrin modulates iron toxicity in a Drosophila model of Friedreich׳s ataxia

“…To perform the interactions in a homogeneous genetic background, we generated a parental strain containing the actin-GAL4, the TM6B TubGAL80 balancer chromosome, and the fhRNAi-2 construct (fhRNAi-2/CyO; actin-GAL4/TM6B TubGAL80). The balancer chromosome (TM6B) encodes the GAL4 repressor GAL80 under the control of the ubiquitous tubulin promoter [49] and therefore, frataxin levels should not be affected in the strain. Indeed, our results showed that the parental strain displays normal frataxin mRNA levels and survival comparable to control strains after hyperoxic insult and on an iron-rich diet (Supplementary Fig.…”