Ongoing transposition in cell culture reveals the phylogeny of diverse Drosophila S2 sublines

Cultured cells are widely used in molecular biology despite poor understanding of how cell line genomes change in vitro over time. Previous work has shown that Drosophila cultured cells have a higher transposable element content than whole flies, but whether this increase in transposable element content resulted from an initial burst of transposition during cell line establishment or ongoing transposition in cell culture remains unclear. Here, we sequenced the genomes of 25 sublines of Drosophila S2 cells and show that transposable element insertions provide abundant markers for the phylogenetic reconstruction of diverse sublines in a model animal cell culture system. DNA copy number evolution across S2 sublines revealed dramatically different patterns of genome organization that support the overall evolutionary history reconstructed using transposable element insertions. Analysis of transposable element insertion site occupancy and ancestral states support a model of ongoing transposition dominated by episodic activity of a small number of retrotransposon families. Our work demonstrates that substantial genome evolution occurs during long-term Drosophila cell culture, which may impact the reproducibility of experiments that do not control for subline identity.     

Identification of insect cell lines and cell‐line cross‐contaminations by nuclear ribosomal ITS sequences

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR‐RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR‐based method used three species‐specific primer sets, Ly‐ITS1/Ly‐ITS2, ITS1‐1/Ld‐ITS1 and Sf9‐F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell‐line cross‐contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell‐line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.     

A proteomics approach to identifying fish cell lines

Fish cell lines are relatively easy to culture and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred. Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2-DE and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. Similar concordance was seen in cells derived from the same tissues than from other tissues within the same species. These profiles have been saved in an electronic databank and could be made available to be used for discerning the origins of the various cell lines evaluated. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines.     

Distribution of antigens in established cell lines of Drosophila melanogaster

Antibodies prepared against two Drosophila cell lines together with antibodies prepared against other Drosophila extracts were used to study the distribution of antigens in 10 cell lines. The results suggest: (1) that cultured cells express differentiated functions; (2) that each cell line, including sub-clones of a clone, is unique; and (3) that the cell lines are derived from the lateral ectoderm.     

The contribution of cell death to medium-released fractions of cell cultures

Summary Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [³H]thymidine and by subsequently measuring the release of label into complete culture medium or serum-and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [³H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the³H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account. This research benefited from use of the Cell Culture Facility supported by National Cancer Institute Grant CA 14733. This research was supported by a Muscular Dystrophy Association Grant to Dr. Heinz Herrmann and by American Cancer Society Grant RDP 8 and was submitted in partial fulfillmant of a Ph.D. degree at the University of Connecticut (T. C. Doetschman).     

Generation of genome-modified Drosophila cell lines using SwAP

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.     

Effect of age on the growth and response of a Drosophila cell line to moulting hormone

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line CI8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of β-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.     

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.     

Purine and pyrimidine salvage in a clonal Drosophila cell line

A new culture medium, ZH1%, for Drosophila cell lines is described in which treatment of cells with the folate antagonist methotrexate (MTX) leads to overt double auxotrophy for both purines and pyrimidines. Using this medium the response of a clonal Drosophila cell line, KcAlo, to various purines and pyrimidines was measured. Guanosine was found to balance the toxicity of adenosine (AR), and vice versa. In addition AR toxicity was shown to be strongly dependent on the pH of the culture medium. Using ZH1% supplemented with MTX + thymidine (TdR) or with MTX + inosine (HR) several purines or pyrimidines, respectively, were tested for their capacity to support cell proliferation. This provided evidence for pathways for the salvage of thymine, TdR, thymidylic acid, adenine (A), AR, and HR. Bromodeoxyuridine and fluorodeoxyuridine are very active and specific TdR analogues inhibiting Drosophila cell proliferation. Of the many purine analogues tested the only potent growth inhibitor with demonstrable pathway specificity (antagonizable by A only) was methylpurine.     

Expansion of human embryonic stem cells: a comparative study

Objectives: Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. Materials and methods: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions – TrypLE? Express, Trypsin‐EDTA, Cell Dissociation Solution and Accutase?– were combined with two application protocols. As reference conditions, manual and bead‐based passaging techniques were used. Results: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. Conclusion: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single‐cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.     

The expression of PS integrins in Drosophila melanogaster imaginal disc cell lines

Summary Drosophila imaginal disc cell lines show a characteristic pattern of aggregation in culture, which appears to be due to cell-cell rather than cell-substrate interactions. We have examined the distribution of PS integrins in wing and leg cell lines, and find that these integrin homologues are expressed preferentially in aggregates. Cell sheets, small cell clumps and chains of cells express antigen at points of cell-cell contact only.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

Transfer of mitochondrial DNA to nuclear genome of cells of passaged cell line of Drosophila virilis

The variability of the chromosomal fragments of the atp6 mitochondrial gene, which is integrated into chromosomal DNA in the lines of flies of different geographic origins and in the passaged cell lines of D. virilis has been analyzed. We did not reveal any nucleotide variability in this DNA marker among the studied fly lines. This result is consistent with the proposition that the D. virilis species is monomorphic. The new fragments of the atp6 gene that are associated with the insertions of the Tv1 retrotransposon and are absent in the fly genome are revealed in the genome of the passaged cell line of D. virilis. This fact is evidence of the activation of the mitochondrial DNA transfer into the nuclear genome of the cells of passaged cell culture.     

Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.     

DNA copy number evolution in Drosophila cell lines

Background

Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.

Results

Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).

Conclusion

Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/gb-2014-15-8-r70) contains supplementary material, which is available to authorized users.     

Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn.