Drosophila Cells in Culture

By Guy Echalier, Norbert Perrimon and Stephanie E Mohr

Drosophila Cells in Culture, Second Edition, includes comprehensive coverage of cell lines, methods for creating cell lines, methods for genome engineering, and the use of cell lines for genome wide rNAi screens. This publication summarizes over thirty years of experience in the handling of in vitro cultured Drosophila cells alongside recent methods and functional screens. Early and experienced researchers studying drosophila in developmental biology, genetics, neuroscience, and across the biological and biomedical sciences will benefit from this expert knowledge.

• Offers full coverage of cell lines and primary cultures
• Provides a go-to resource for methods and studies completed with drosophila cells in culture
• Presents a wide spectrum of experimental techniques

• Language: English
• Publisher: Academic Press
• Release date: Nov 30, 2017
• ISBN: 9780128097045

Guy Echalier

Guy Echalier is an esteemed expert in the research of Drosophilia, having over thirty years experience handling in vitro cultured Drosophilia cells.

Book preview

Echalier

Introduction to the Second Edition

The 120-megabase euchromatic portion of the Drosophila melanogaster genome has been sequenced…. This sequence may be ‘the Rosetta stone’ for deciphering the human genome.

(Kornberg and Krasnow, 2000)

The fact that this second edition of Drosophila Cells in Culture appears 20 years after the first edition was published reflects the enduring value of the original book and the state of the field. For a long time, the original Drosophila cell cultures, such as Kc and S2 cultured cell lines, have been the go-to choices for Drosophila cell studies, even as new applications, such as large-scale genomics and transcriptomics analyses, were applied. More recent developments are now shifting the field, however, by providing researchers with the ability to develop new cell lines and genotypes with increasing ease and precision.

We had two goals for this second edition of the book. First, we wished to preserve the historical background and classical methods described in the first edition. To this end, we begin with a general introduction and five chapters that describe how the original Drosophila cell lines were generated, how media formulations were developed, and other foundational information. In Chapters 6–9, we shift focus to more contemporary approaches. In these chapters, we aim to present background information and protocols for technologies developed subsequent to the first edition, including the isolation of new cultured cells using an oncogene approach, the application of large-scale functional genomics screening to Drosophila cells, and new approaches to gene perturbation. In the final chapter, we present a compendium of cell lines, protocols, plasmid vectors, and data sets available as community resources.

–Guy Echalier, Norbert Perrimon, and Stephanie E. Mohr

General Introduction

Guy Echalier

Abstract

Drosophila melanogaster offers many advantages for experimental study and has been used in research for more than 100 years. With a DNA content 50 times greater than that of Escherichia coli bacteria and 30 times smaller than that of mammals, it offers a balance of complexity and simplicity. Functional and comparative genomic studies have also revealed a high degree of gene conservation between flies and other species. The use of cultured cells offers a number of advantages for experimentation. Many Drosophila cultured cell lines, as well as media conditions, have been established, facilitating a wealth of studies.

Keywords

Drosophila cell culture; insect cell culture; Drosophila genetics; gene conservation; historical perspective

The 120-megabase euchromatic portion of the Drosophila melanogaster genome has been sequenced…. This sequence may be the Rosetta stone for deciphering the human genome.

(Kornberg and Krasnow, 2000)

I A Century of Drosophila Genetic Research

Around 1910, the small vinegar fly—among some five other tested animals—was adopted by T.H. Morgan and his group of early American geneticists, because of its ease of handling, small size (that means the possibility of breeding hundreds of individuals in small bottles), cheap food (simple pap of maize, a basic traditional American food!), rapid generation time (12 days for the succession of egg, maggot, pupa, and imago), and the simplicity of its chromosome complement (four pairs of chromosomes easily identifiable + existence of giant polytene chromosomes).¹

For more than a century, generations of investigators, throughout the world, have accumulated vast volumes of information on the biology of this organism, which has no rival amongst higher eukaryotes.

II Transient Eclipse but Unavoidable Come Back of Drosophila in Avant-Garde of Biology

The fundamental relationship between nucleic acids and protein synthesis—after the brilliant unraveling of the bihelicoidal structure of DNA by Watson and Crick (1953), and then the discovery of messenger RNA—had, for technical reasons, to be established on the small genomes of prokaryotes, such as bacteria (essentially Escherichia coli) and viruses. Thus, during some 20 or 30 years of these transformative times, Drosophila knew a relative eclipse (Fig. I.1).

Figure I.1 Stained metaphases of Drosophila Kc line cells. Metaphase plate, after blocking with colchicine and staining with orcein, clearly shows the characteristic diploid karyotype of Drosophila melanogaster. Reprinted from Echalier, G., Ohanessian, A., 1969. Isolation, in tissue culture, of Drosophila melangaster cell lines. C.R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 268 (13), 1771–1773, Copyright (1969), with permission from Elsevier.

However, due to lightening technical progresses and fascinating experimental results of this new discipline—rapidly baptized Molecular Biology—the time rapidly came to tackle the more complex genomes of highest Eukaryotes; and, for this new research stage, Drosophila offered unrivaled assets: the unusual small size of its genome favors biochemical approaches: the 0.18 pg of nuclear DNA per haploid genome of a Drosophila cell is approximately halfway between the genetic materials of bacteria and mammalian cells. Broadly speaking, its DNA content is only 50 times higher than that of E. coli and 30 times smaller than that of mammals (6 to 7 pg in Homo sapiens).

III Brief Historical Story of Drosophila Cell Culture

By chance, Drosophila cell cultures have been actively developed during this interval:

A few years after the epochal in vitro cultures of the amphibian embryonic neural tube by Harrison (1907), whereby he demonstrated the growth of axons from the soma of neuroblasts (thus providing direct proof of the structural unity of the neuron); then, Goldschmidt (1915) reported silkworm spermatogenesis in similar hanging drop cultures. This latter work may be considered as the birth of invertebrate tissue culture.

A decisive breakthrough in cell culture techniques was made when, during the 1950s, cell monolayers were grown in liquid culture media. Insect cell cultures needed about 10 years to catch up with vertebrate cell culture technics, which is not surprising since the number of laboratories involved in the field was much smaller.

Moreover, it is important to emphasize that no significant success was achieved until the specific biochemical features of insect body fluids were taken into consideration. Thus, a detailed analysis of Bombyx larval hemolymph resulted in the initial designing by the Wyatts of an original culture medium in which ovarian sheath cells were able to migrate and multiply (Wyatt, 1956). Finally, a few slight modifications in this recipe allowed T. Grace, in 1962, to grow in vitro the first permanent insect cell lines, from ovaries of a large lepidopteran, Antherea (Grace, 1962).

As for Drosophila tissues, early attempts of culture were focused on imaginal discs and their capacity for morphogenesis in vitro. Among precursor works, the numerous short notes by Kuroda (see bibliography, Chapter 4: Cells or Tissues in Course of Differentiation), the limited successes of Cunningham (1961) and Hanly (1961) with the much more substantial papers by Schneider (1963, 1964, 1966) should be remembered.

Finally, the first bona fide primary embryonic cell cultures (Echalier et al., 1965) of Drosophila, and principally, the first permanent cell lines [called Kc and Ca, from the designations of their initial culture flask (Fig. I.2)].

Figure I.2 Kc cells: observe their clear nuclei with single dark nucleolus. Kc cell line. Phase-contrast microscopy: in the large and clear nucleus, observe the single dark nucleolus. Reprinted from Echalier, G., 1971. Established diploid cell lines of Drosophila melanogaster as potential material for the study of genetics of somatic cells. In: Weiss, E. (ed.), Arthropod Cell Cultures and Their Application to the Study of Viruses. Current Topics in Microbiology and Immunology (Ergebnisse der Mikrobiologie und Immunitätsforschung), vol. 55. Springer, Berlin, Heidelberg, with permission of Springer.

Shortly after, the Russian scientists, Gvozdev and Kakpakov (1970) published similar results.

Note: An extremely interesting experiment, for demonstrating the value of such lines, consisted to transplant a few nuclei into a very young egg and to verify that they were perfectly incorporated into the formation of the embryo.

• Among the multiple Drosophila continuous cell lines which have been established since those first successes—i.e., along the last half century—we must imperatively quote also the S2–S3 lines of Schneider (1972), which today are among those widely used (see Chapter 3: Continuous Drosophila Cell Lines Established In Vitro).

IV Current Prospects

A Culture Media and Techniques Would Need to be Significantly Improved

Let us emphasize the exceptional interest of such experimental material for biochemical research: availability of theoretically unlimited amount of a homogeneous (after a recent cloning) cellular material; existence of multiple new cell lines from well characterized Drosophila mutants; possibility of cell transformation by transfection, and thus production and recovery of interesting gene products.

However, as we discuss in Chapter 3, Continuous Drosophila Cell Lines Established In Vitro, most, if not all cells of the many cell lines of Drosophila established in vitro prior to year 2000 exhibited—in spite of their difference in tissue of origins—a very banal morphology, losing their specific structures and physiological characteristics; as if cells had been selected to concentrate on basic functions, compatible with rapid growth and proliferation. Such a choice might be an advantage for molecular analyses; however, it will arrive a time when the specific mechanisms of differentiation—a crucial characteristic of higher organisms—will no longer be neglected.

Improving culture techniques remains, indeed, time-consuming and a moderately gratifying work! So, since pioneering times, very few people have taken care of improving the basic culture media and methods.

However, as a decisive progress in the establishment in vitro of cell lines from a definite tissue origin, we must bring to light the efficient genetic RasV12 method designed by Simcox et al. (2008), where they designed a simple and efficient method to establish cell lines based on the expression of an oncogene.

Moreover, decisive progress may be reasonably expected from our present deep studies—and much better understanding—of the complex interferences between multiple hormones and growth factors, during insects cell growth, multiplication, and differentiation (see Chapter 5: Hormonal Control of Development of Insects: Implications in Organ or Cell Cultures of Drosophila).

B A Divine Surprise: Discovery of Orthologous Genes Between Drosophila and Homo Sapiens

After the complete sequencing of the nuclear genome of Drosophila melanogaster, it was discovered that some 60% of its genes have some homologous base sequence with human genes!

Sometimes, such genes may have acquired distinct functions during evolution but, frequently enough, they were found to be still involved in quite similar morphological or physiological activity and to have retained the same function.

It is obvious that, if such genes have been kept during the several hundred millions of years which, according to evolutionists, would have separated the development of flies and humans, it may be assumed that they do concern basic functions of life! Another proof of their peculiar importance is the finding that up to 75 per 100 of the human genes which are, more or less, implicated in some pathological heredity, would belong to this category! It is easy to imagine the fabulous experimental fields that are opening up behind this discovery, in particular, for human pathology and therapeutics.

As a rapid Conclusion, Drosophila cells have taken, today, the prominent role played by bacteria and viruses in the development of Molecular Biology. In an approximate abridgement, we might say that they have become, for avant-garde Biology, the Escherichia coli of this beginning century!

References

1. Cunningham, I., 1961. Studies on the Maintenance and Growth of Insect Tissue and Cells In Vitro. Master of Science, University of Edinburgh.

2. Echalier G, Ohanessian A. Isolation, in tissue culture, of Drosophila melanogaster cell lines. C.R Acad Sci Hebd Seances Acad Sci D. 1969;268:1771–1773.

3. Echalier G, Ohanessian A, Brun G. Primary cultures of embryonal cells of Drosophila melanogaster (dipterous insect). C.R Acad Sci Hebd Seances Acad Sci D. 1965;261:3211–3213.

4. Goldschmidt R. Some experiments on spermatogenesis in vitro. Proc Natl Acad Sci USA. 1915;1:220–222.

5. Grace TD. Establishment of four strains of cells from insect tissues grown in vitro. Nature. 1962;195:788–789.

6. Gvozdev VA, Kakpakov VT. Establishment of female embryonic cell sublines of Drosophila melanogaster in vitro. Drosophila Inf Serv. 1970;45:110.

7. Hanly, E.W.I., 1961. Tissue culture of Drosophila. Pteridines and eye pigmentation in Drosophila melanogaster. Ph.D., The University of Texas at Austin.

8. Harrison RG, Greenman MJ, Mall FP, Jackson CM. Observations of the living developing nerve fiber. Anat Rec. 1907;1:116–128.

9. Kornberg TB, Krasnow MA. The Drosophila genome sequence: implications for biology and medicine. Science. 2000;287:2218–2220.

10. Schneider I. In vitro culture of Drosophila organs and tissues (Abstract). Genetics. 1963;48:908.

11. Schneider I. Differentiation of larval Drosophila eye-antennal discs in vitro. J Exp Zool. 1964;156:91–103.

12. Schneider I. Histology of larval eye-antennal disks and cephalic ganglia of Drosophila cultured in vitro. J Embryol Exp Morphol. 1966;15:271–279.

13. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972;27:353–365.

14. Simcox A, Mitra S, Truesdell S, et al. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes. PLoS Genet. 2008;4:e1000142.

15. Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 1953;171:737–738.

16. Wyatt SS. Culture in vitro of tissue from the silkworm, Bombyx mori L. J Gen Physiol. 1956;39:841–852.

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¹Very few organisms possess such polytene chromosomes; on this short list, is also a plant, the pea, Mendel’s experimental material. When, in the 1930s, such chromosomes were rediscovered in Diptera such as Drosophila, N.W. Timofeef-Ressovsky, a renowned Russian geneticist, used to say, as a joke, that the presence of giant chromosomes in these two major genetic materials was, according to him, a proof of the existence of God!

Part I

Historical Perspectives and Classical Methods

Outline

Chapter 1 Composition of the Body Fluid of Drosophila and the Design of Culture Media for Drosophila Cells

Chapter 2 Primary Cell Cultures of Drosophila Embryonic Cells

Chapter 3 Continuous Drosophila Cell Lines Established In Vitro

Chapter 4 Cells or Tissues in Course of Differentiation

Chapter 5 Hormonal Control of Development of Insects: Implications in Organ or Cell Cultures of Drosophila

Chapter 1

Composition of the Body Fluid of Drosophila and the Design of Culture Media for Drosophila Cells

Guy Echalier,    Center National de la Recherche Scientifique, Universite Pierre et Marie Curie, Paris, France

Abstract

Successful cell culture depends on successful formulation of appropriate cell culture media and growth conditions. Defining the milieu interieur of Drosophila hemolymph provided a starting point for defining appropriate osmolarity, pH, ionic content, amino acids, and sugars, and so on to support Drosophila cell culture. Supplementation with sources of vitamins, such as from yeast extract, serum, and hormones or growth factors further influence growth of cells in culture. This work has resulted in definition of a number of standard media recipes that are presented and discussed in the chapter along with background information.

Keywords

Drosophila cell culture; cell culture media; culture media formulation; insect physiology; osmolarity; Drosophila extracts

Chapter Outline

Introduction 4

Milieu Interieur of Drosophila Larva 4

I. Biochemical Data About the Milieu Interieur of Drosophila 5

A. Osmotic Pressure 5

B. pH 6

C. Inorganic Ions 7

D. Amino Acid Pool 9

E. Organic Acid 13

F. Sugars 13

II. Main Available Culture Media for Drosophila Cells 13

A. Echalier and Ohanessian’s D22 Medium 14

B. Gvozdev and Kakpakov’s C-15 or S-15 Medium 16

C. Schneider’s Medium 18

D. Medium M3: Shields and Sang’s (1977) + Cross and Sang (1978) 20

E. Wyss’s Medium ZW 22

F. Robb’s Medium R-14 25

III. General Discussion About the Characteristics and Main Components of Culture Media for Tissues or Cells of Drosophila 28

A. Osmolarity 29

B. pH and Buffers 30

C. Major Ions 30

D. Amino Acids 32

E. Sugars 34

F. Vitamins—Yeast Extracts 35

G. Serum Supplementation 37

H. Hormones and Growth Factors 38

I. Drosophila Fly and Egg Extracts 39

J. Antibiotics 40

IV. Prospective Conclusions 40

References 41

Appendix 1.A (as relates to Table 1.5) 44

Appendix 1.B Stock Solution of Grace’s Vitamins 45

Introduction

When an artificial culture medium for growing cells outside the organism is being designed, it seems logical to try to copy, as closely as possible, the natural biological fluid in which the cells are immersed in situ.

Experience has proved, however, that such an imitative approach (Waymouth, 1965) for in vitro cultures should always be tempered with a large dose of empiricism.

Thus, the composition of successful culture media is always the result of a compromise (Paul, 1973).

Milieu Interieur of Drosophila Larva

Only comparative physiologists fully realize how markedly the composition of the milieu intérieur (Claude Bernard) of most other zoological classes—and particularly of insects—differs from that of blood plasmas of higher vertebrates. Yet, it must be remembered that the chemical features common to mammalian sera have inspired all the classical physiological solutions used for handling tissues or the cells, such as the Ringer–Locke solution and its numerous derivatives. Therefore, none of them, or culture media inspired from them, could be ideally suited for culturing invertebrate tissues.

Among insects, the great diversity of the distinct orders should also not be ignored, since there are considerable differences in the composition of their body fluids, from one family to another and even among the species of the same genus! This might partly depend on specific biological habits and feeding but seems to be mainly a question of taxonomy and the revelation of evolutionary tendencies (see review by Florkin and Jeuniaux, 1964). Moreover, within the same species, certain biochemical characteristics can greatly vary from one developmental stage to another. For the Dipteran Drosophila, for instance, it is easy to understand that its larval instars—maggots living in a semiliquid paste—are completely different, in their structure and mode of life, from the aerial flies that will arise during their metamorphosis.

These facts should all be considered when one is elaborating an adequate culture medium.

Because of its importance in genetics and developmental biology, it is not surprising that we have at our disposal quite an important mass of information about the milieu intérieur of Drosophila melanogaster. But for practical reasons—since it is much more difficult to draw off enough fluid from adult flies—most of these analytical data refer to the hemolymph of the late third larval instar.

I Biochemical Data About the Milieu Interieur of Drosophila

A Osmotic Pressure

The osmotic pressure of the hemolymph of D. melanogaster third larval instar is significantly higher than the 300 mOsm figure that characterizes uniformly higher vertebrate plasmas. It was measured to be close to 360 mOsm (see Table 1.1), which corresponds to a sodium chloride solution of 10.5 g/L (instead of the classical 8.5/L NaCl solution that is assumed to be isotonic for mammalian cells). This value might, however, vary slightly, according to the environmental conditions encountered (Croghan and Lockwood, 1960).

Table 1.1

Osmotic Pressure, Drosophila Third Larvae Hemolymph

Begg, M. 1955. Osmotic pressure of Drosophila larval hemolymph. Drosophila Inf. Serv., 29, 105.

Begg, M., Cruickshank, W. J. 1963. A partial analysis of Drosophila larval hæmolymph. Proc. R. Soc. Edinb., 68, 215–236.

Croghan, P. C., Lockwood, A. P. M. 1960. The composition of the hemolymph of the larva of Drosophila melanogaster. J. Exp. Biol., 37, 339–343.

Zwicky, K. 1954. Osmoregulatorische Reaktionen der Larve von Drosophila rnelanogaster. Z. Verg. Physiol., 36, 367–390.

aOsmolarity expressed as mmol dissolved ions per liter.

In contrast with the situation in vertebrates where Cl− and Na+ ions are largely predominant, ionized salts of insect body fluids account for a relatively small proportion of the total osmotic pressure.

The main osmotic effectors are instead organic molecules and especially amino acids. For instance, in Drosophila larval hemolymph, amino acids, and other ninhydrin-positive substances, might be responsible for one-third (Zwicky, 1954), or even 40% (Begg and Cruickshank, 1963) of the total