Natural Variation in Drosophila Behavior - Reed College This article provides the basic methods for the laboratory culture of D. melanogaster. Jianwu Bai. We STRONGLY encourage individual users to experiment with the temperature and duration of wet-yeast incubation and empirically determine the optimal incubation conditions for their experiments. and N.P. Lighting up mRNA localization in, Airoldi SJ, McLean PF, Shimada Y, Cooley L. Intercellular protein movement in syncytial, McLean PF, Cooley L. Protein equilibration through somatic ring canals in, Osterfield M, Du X, Schpbach T, Wieschaus E, Shvartsman SY. 7The combination of CY2-Gal4 [51] and UAS-GFP::Moesin [52] can be maintained stably in a strain and allows visualization of follicle-cell outlines and actin dynamics. If the user needs to use supplemented Schneiders medium for culturing and the cost of the medium is a concern, then the ovaries can be removed from the fly in non-supplemented Schneiders medium, and the ovary pair can then be transferred to a small volume of supplemented Schneiders medium in the second dissecting dish. The size of the blanket can be altered to accommodate different sizes of culture dish and washer, or to satisfy the preference of the user. Shields, G. & Sang, J. Characteristics of five cell types appearing during in vitro culture of embryonic material from Drosophila melanogaster . In Vitro 8, 712 (1972). CAS 8600 Rockville Pike Thomas Hunt Morgan and the historical frame 3. This allows flies to be identified and sorted for experimental work. 23, 5369 (1970). Minimal laser exposure and careful tweaking of imaging parameters are required when using this strain. PubMedGoogle Scholar. The intelligent culture of Drosophila requires a basic understanding of the Proc. PloS. 69, 161173 (1971). Modified Graces medium or Schneiders medium are used alone or with minimal supplementation, depending on the context, and are therefore preferable if complete knowledge of chemical composition is not essential. (1992) The Making of a FlyThe Genetics of Animal Design. Notes also indicate where the protocol can be altered to facilitate culturing of different egg-chamber stages or for use with an upright confocal microscope system. A chronological summary of 40 years of live-imaging efforts using the D. melanogaster ovary, concentrating on fluorescent marker(s) and imaging system(s) used, as well as the time interval(s) between data acquisition. Steinbrink, S. & Boutros, M. RNAi screening in cultured Drosophila cells. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 30Damage to egg chambers during dissection (e.g., breaking, tearing, piercing) can perturb aspects of egg chamber development, and such damage is usually visible under wide-field conditions (e.g., bright field, fluorescence) at 10X or 20X, or even under the stereomicroscope. Natl. The more ovaries that are dissected, the longer it will take to sort through the ovaries and isolate egg chambers of the desired stage. Culture of Drosophila: The Laboratory Setup A Balbiani body and the fusome mediate mitochondrial inheritance during, Spracklen AJ, Fagan TN, Lovander KE, Tootle TL. C illustrates how a brass washer, which is slightly smaller than the culturing well at the bottom of the culturing dish, is first carefully lowered into the culturing medium so that it is centered over the egg chambers under the tissue (left), and then VERY gently released so that no egg chambers are crushed (right). Adams, M.D. Abstract. WebDrosophila melanogaster, more commonly known as fruit fly, is one of the most widely used organisms to study genetics. Overview chapter: non-standard format - University of Dayton CAS Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Greenspan, R. J. The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Drosophila 10 male Drosophila and 10 female Drosophila are shifted into the bottle which contains new medium/ substrate and the bottle is closed with the cotton. A protocol for culturing. Biol. However, incubation with a membrane marker, such as FM 4-64, or a nuclear stain, such as DAPI or Hoechst 33342, can be a useful technique for detecting subtle egg-chamber damage [48]. Prasad M, Jang AC, Starz-Gaiano M, Melani M, Montell DJ. 36If the brass washer is not held horizontally, it will be very difficult to move it into place without risking crushing the egg chambers. Still images are adapted from supplementary material in [47] with permission from Elsevier. Clemens, J. et al. Methods in Molecular Biology, vol 420. Spatiotemporal control of small GTPases with light using the LOV domain. Ovary dissection, egg chamber isolation, and transfer to the culturing device should then be performed as quickly as possible, and the culturing device should be immediately placed on a temperature-controlled microscope stage. If culturing and live imaging must be performed at a temperature other than room temperature, we use a temperature-controlled stage that is compatible with 35-mm culture dishes. Note that in the side view the culturing dish lid has been replaced, to prevent evaporation of culturing medium. 1405. These culture dishes require the use of an adjustable stage, or stage adaptor, and are also compatible with certain temperature-controlled stages. During S6S9 (24 hours) the egg chamber elongates along the AP axis, the nurse cells continue their endoreplication cycles (>500C), the oocyte expands significantly compared to its interconnected nurse-cell siblings (due in large part to uptake of yolk proteins), the follicle cells cease dividing and undergo endoreplication, the vitelline membrane begins to form, and the morphogenetic events of the follicular epithelium begin (i.e., the columnarization of posterior follicle cells contacting the oocyte, the flattening of follicle cells over the nurse cells, and the migration of the border cells). The Drosophila female abdomen is divided into 8 segments (A1-A8, anterior to posterior), and each of these segments is covered in a cuticular plate called a sclerite. If supplements to the culturing medium meant that using a large volume of medium is too expensive, the device could utilize a smaller volume of conditioned medium in a humidity chamber (see [19]). et al. This in turn has made fruit flies an attractive system for many other biomedical researchers who have otherwise no background in fly biology. J.B. and K.S. The inner diameter of this washer is 4 mm, a diameter that provides just enough space to cluster the egg chambers in its center without crushing them. It is being Note that the image is noisier because different imaging settings had to be used to detect the weaker signal. In the germline, cytoplasmic streaming within the oocyte was first documented and imaged in egg chambers cultured in Robbs medium [7]. Drosophila egg chambers on an inverted confocal microscope. First, their ovaries respond rapidly to the presence of wet yeast paste, which stimulates egg chamber production. Spinning disc confocals will usually have lower resolution than scanning confocals, but allow for much faster Z-stack acquisition, more frequent time points and/or more recovery time between Z-stacks, and overall lower risk of phototoxicity. The intelligent culture of Drosophila requires a basic understanding of the life cycle of this fly. For this reason, we begin by describing the life cycle of Drosophila and then present information for setting up a fly laboratory. External, adjustable white-light illumination (. *The materials described below are for the dissection, culturing, and live imaging of late-stage 20If the desired egg chambers for culturing are S10BS11, as they are for our studies, the ovaries will be very large and the abdomens of the females will be swollen. As long as the tissue of interest is not directly exposed to halocarbon oil (e.g., nurse cells, oocyte, migrating border cells), this medium can support normal development and may be preferred due to its amenable characteristics for microscopy ([13], [14], [15]). Perrimon, N. & Mathey-Prevota, B. 29If the user opts to use only two dissecting dishes, the pipette manipulator should be used to remove all extra ovarioles, ovariole remnants, and other material from the second dissecting dish, as well as the majority, but not all, of the Schneiders medium. 14We use VWR 82003-822 Light-Duty Tissue Wipers for creating our egg chamber immobilization blankets, and we find that contact with this material has no adverse effects on egg chamber development. & Perrimon, N. Approaches to identify genes involved in Drosophila embryonic CNS development. This setup provides a more buffered, stable culturing environment than previously published methods by using a larger volume of culture media, but the setup is also compatible with small volumes. Get the most important science stories of the day, free in your inbox. The culturing of late-stage egg chambers (i.e., S10+) is possible in unaltered, sterile Schneiders medium ([20], [21]) or in Schneiders medium supplemented with fetal bovine serum and, to suppress bacterial growth, penicillin-streptomycin ([34]). WebThe use of Drosophila cell culture provides: (1) The rapid testing of potential in vivo transgenic constructs for proper biochemical and cellular functions before creating Alternatively, the user can gently tug on the edges of the immobilization blanket with the forceps while the brass washer is in place to increase the tension on the egg chambers. Illustrated instructions for assembling an apparatus for culturing and live imaging D. melanogaster egg chambers. Culture of Drosophila While this liberation can be a fortunate situation, these egg chambers should be examined carefully to make sure the forceps have not damaged them before using them for live imaging (see Shields, G., Dubendorfer, A. There are four distinct stages in the life of the fruit fly: egg, larva, pupa and adult. The goal of the dissector should be to dissect ovaries and isolate egg chambers as quickly and cleanly as possible, so the number of females necessary for a culturing experiment will ultimately depend on the preference and skill of the dissector and on the strain being dissected.
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