In vitro culture of drosophila melanogaster embryonic cells
01 Nov 1970-In Vitro Cellular & Developmental Biology – Plant (Springer-Verlag)-Vol. 6, Iss: 3, pp 162-172
TL;DR: A new culture medium for cells from Drosophila embryos has been designed that has an osmotic pressure equivalent to 10.5 g per liter of NaCl, Na+: K+ ratio of 1.4, and very low Cl−.
Abstract: A new culture medium for cells fromDrosophila embryos has been designed. It has an osmotic pressure equivalent to 10.5 g per liter of NaCl (pH 6.7), Na+: K+ ratio of 1.4, and very low Cl−. Glycine and glutamic acid are high. Cells grow well for several weeks in this medium at 25–27°C and some cultures persist for months and establish euploid lines.
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TL;DR: Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells and a family of attachment sites related by hybridization to those of the hsp70 genes was discovered.
1,017 citations
TL;DR: A high-throughput RNA-interference screen of 19,470 double-stranded RNAs in cultured cells to characterize the function of nearly all predicted Drosophila genes in cell growth and viability found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles.
Abstract: A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.
787 citations
TL;DR: Using RNAi, genes that influence cytoskeletal organization and morphology in two distinct cell types are identified and proposed to propose similar functions for previously uncharacterized genes.
Abstract: Background: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. Results: We adapted existing RNAi technology in Drosophila cell culture for use in highthroughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. Conclusions: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.
453 citations
TL;DR: A user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users are provided.
Abstract: RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.
396 citations
TL;DR: This study examines pausing on hsp70 and two of the small heat shock genes at high resolution using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts, which provides precise information on the distribution of RNA polymerase within each transcription unit.
Abstract: The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.
371 citations
References
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821 citations
"In vitro culture of drosophila mela..." refers methods in this paper
...Vitamins Yeast extract Vitamins as in Grace's culture medium for Antheraea cells (37) 5....
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...In the absence of quantitative information on vitamins in Drosophila hemolymph, we have used the concentrations suggested by Grace for the culture of lepidopteran cells (37)....
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TL;DR: Hemolymph was collected for analysis from the silkworm in a series of developmental stages ranging from the second molt to the late pupa, indicating the presence of low molecular weight sugar derivatives and the mean pH of larval hemolymph after collection was found to be 6.45; in vivo values may be slightly lower.
Abstract: α,α-Trehalose, a sugar previously regarded as a product characteristic of certain lower plants, has been identified as a major blood sugar of insects. Trehalose has been isolated in pure form from the blood of pupae of the silk moth, Telea polyphemus , and has been recognized chromatographically in all the insects examined, which comprise 10 species belonging to 5 different orders. Trehalose has been determined quantitatively with anthrone after either chromatographic separation or chemical degradation of other sugars. In larvae and pupae of 4 species of Lepidoptera it ranges from 0.2 to 1.5 gm. per 100 ml. of blood and makes up over 90 per cent of the blood sugar; in larvae of a sawfly, about 80 per cent of the blood sugar is trehalose. In Bombyx mori and Platysamia cecropia , the pupal blood trehalose level is about half that in the mature larva, suggesting utilization of trehalose for glycogen synthesis during pupation. Small amounts of glucose and apparent glycogen are also present in the plasma of these insects. In Bombyx larval plasma there is also 0.04 to 0.12 gm. per 100 ml. of glucose-6-phosphate and smaller amounts of an apparent ketose phosphate.
494 citations
TL;DR: α,α-Trehalose, a sugar previously regarded as a product characteristic of certain lower plants, has been identified as a major blood sugar of insects and determined quantitatively with anthrone after either chromatographic separation or chemical degradation of other sugars.
Abstract: α,α-Trehalose, a sugar previously regarded as a product characteristic of certain lower plants, has been identified as a major blood sugar of insects. Trehalose has been isolated in pure form from the blood of pupae of the silk moth, Telea polyphemus, and has been recognized chromatographically in all the insects examined, which comprise 10 species belonging to 5 different orders. Trehalose has been determined quantitatively with anthrone after either chromatographic separation or chemical degradation of other sugars. In larvae and pupae of 4 species of Lepidoptera it ranges from 0.2 to 1.5 gm. per 100 ml. of blood and makes up over 90 per cent of the blood sugar; in larvae of a sawfly, about 80 per cent of the blood sugar is trehalose. In Bombyx mori and Platysamia cecropia, the pupal blood trehalose level is about half that in the mature larva, suggesting utilization of trehalose for glycogen synthesis during pupation. Small amounts of glucose and apparent glycogen are also present in the plasma of these insects. In Bombyx larval plasma there is also 0.04 to 0.12 gm. per 100 ml. of glucose-6-phosphate and smaller amounts of an apparent ketose phosphate.
320 citations
"In vitro culture of drosophila mela..." refers methods in this paper
...As was previously shown by Grace and Brzostowski (33) for lepidopteran cells, one can use glucose in the culture medium even though the predominent carbohydrate in insect hemolymph is trehalose (28, 38), the hydrolysis of which yields 2 moles of glucose....
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TL;DR: The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.
Abstract: A phosphate-buffered saline and a chemically defined synthetic medium for in vitro maintenance of imaginal discs of Drosophila melanogaster were developed. The composition of the chemically defined medium was varied in order to optimize the incorporation of tritiated uridine into RNA and tritiated amino acids into acid-insoluble protein. The optimal ranges obtained were: pH, 6.75–7.35; osmolarity, 285–345 milliosmoles/liter; sodium concentration, 40–60 mM/liter; potassium concentration, 40–60 mM/liter; magnesium concentration, 0.5–3.5 mM/liter; calcium concentration, 0.3–1.5 mM/liter; and inorganic phosphate concentration, 1.5–4.0 mM/liter. The phosphate-buffered saline is superior to a commonly used insect Ringer solution in maintaining total RNA and acid-insoluble protein synthesis in culture. The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.
293 citations
"In vitro culture of drosophila mela..." refers result in this paper
...While several lines of mosquito cells have been established in the last few years (Aedes aegypti, Aedes albopictus (1-3); Anopheles stephensi (4)), the published results with Drosophila material (5-22) were, until recently, very limited and rather disappointing....
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238 citations
"In vitro culture of drosophila mela..." refers result in this paper
...While several lines of mosquito cells have been established in the last few years (Aedes aegypti, Aedes albopictus (1-3); Anopheles stephensi (4)), the published results with Drosophila material (5-22) were, until recently, very limited and rather disappointing....
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