Many studies concerned with the function of a miRNA will involve either a knockout or driven overexpression of its gene. For this phase of screening, we are opting for the former. This is to afford inclusivity of as many miRNAs as possible, as overexpression-enabling lines are available in greater numbers than knockdown-enabling lines. Overexpression constructs in this case are comprised of miRNA genes tethered downstream to a UAS sequence. Longhand genotypes, as typically seen on the stock centre website, depicts the type of plasmid used to introduce the construct into the Drosophila genome, as well as the chromosomal point integration is expected to occur. Balancer phenotypes are described here as well. It is important to hold them to note as individuals from the F1 generation overexpressing a miRNA of interest are often selected through against-phenotype screening. In theory these flies will produce GAL4 proteins in driver-determined tissues. As explained previously, the GAL4-UAS interaction will activate the miRNA gene, and the effects of its excessive presence within cells can subsequently be defined.
Our screening involves three types of drivers. Each was chosen on the basis of their reported specificity, and how we could exploit their expression patterns in order to observe cytoophidia changes within ovaries. The first type drives expression in follicle cells; the second is for germline cells (nos); and the third is ubiquitously expressed i.e. will be active in all cell types (Act5C). The employment of two follicle cell drivers should lend further credibility where commonly shared modifications to cytoophidia shape, size, and/or length are observed. The utilization of nos should allow for better visibility of miRNA-caused phenotypes in germline cells, where cytoophidia are much larger in comparison to how they appear in follicle cells. Although the overexpression of a majority of known Drosophila miRNAs under Act5c are known to be lethal, the inclusion of a ubiquitous promoter in this experimental design is nonetheless important: as their effects on CTPsyn in general is relatively unknown, small-scale activation of miRNA-expression as afforded by cell-specific drivers such as T155 and nos may be inadequate to display the changes they could truly impose upon the cytoophidia-structure. Driving expression under Act5c is therefore a useful back-up measure, as it should highlight the modifications to cytoophidia otherwise obscured by inadequate gross over-generation of the candidate miRNA. We have now identified four miRNAs as prime candidates. Two shortens cytoophidia, whereas the other two lengthens them. Questions which beg addressing herewith are why, which, and how: why does miRNA overexpression affect cytoophidia in such a way? Which mechanisms and pathways are regulated by this particular miRNA, and how does this relate back to CTPsyn compartmentation? Are they direct regulators of the protein, or do they instead elicit its upstream effectors?
In this chapter, we aim to provide answers by in silico target prediction, followed by traditional validation methods, both in vivo and in vitro. Multiple target prediction tools are freely available online (Exiqon). Each utilizes its own unique algorithm to either predict target mRNAs of a miRNA, or miRNAs which could bind the gene of interest. For D. melanogaster, only one has been configured to specially include species-specific phylogenetic considerations. For this reason, TargetScanFly shall be employed for our in silico prediction purposes.
Several strategies are to be applied in in vivo analysis. In the previous chapter, we showed how cytoophidia alterations within a nurse cell can be caused by miRNA overexpression in its encapsulating follicle cells. Here, we are proceeding with the knocking-down of candidate miRNAs. For Drosophila, the knockdown of a miRNA gene is possible through the application of ‘sponging’, whereby constructs carrying multiple copies of the perfect complementary sequence of an active miRNA are introduced into the cell, effectively diverting it away from its natural endogenous targets. However, elimination of a miRNA is often difficult to substantiate, and even when achieved could be developmentally lethal. Furthermore, shared functionality is widespread amongst miRNAs; where sponges of a particular one is in effect, endogenous expression of another will likely compensate for its absence. Any effects observed thus cannot be attributed to the elimination of the miRNA of interest, rendering the whole exercise void. For this reason, knockout of individual miRNAs by sponging was bypassed in favor of overexpression as the preliminary method of shortlisting candidate miRNAs.
This is achievable through crosses involving lines carrying miRNA-sponges; these should eliminate candidates, enabling us to determine whether their absence would have effects opposing their overexpression. Cytoophidia localization patterns, distribution, and traits have furthermore been extensively described in D. melanogaster testes. The utilization of Act5c should thusly allow us to examine the effects of candidate miRNA overexpression in these tissues as well.
In vitro validation is to be wholly conducted in cell culture. The usage of fly cell lines as a model system towards configuring pathways and processes has been common practice for over forty years. More than a hundred lines are available in order to cater for a wide variation of experimental needs. Most drosophilid cell lines are either embryonic or larval in origin. Schneider-2 or S2 and Kakpakov-c or Kc are the two most often utilized, as their behavior and characteristics are well studied and thus recorded.
Studies conducted with these lines have contributed to our understanding of blood cell immunity in the species. One of the greatest points of exploitation of S2 and Kc are the fact that the in vivo population of cells they mimic most closely in terms of behavior is hematopoietic cells. By taking advantage of this peculiarity we now know the genetic factors driving antibacterial activity upon initial exposure to such an antigen, as well as how residual bodies of bacteria and dying cells are eliminated from healthy counterparts.
With respect to developmental biology, the usage of Drosophila cell lines has led to the delineation of several growth factor signaling pathways. One such example is how the relationship between Daughterless and Nautilus was determined to be myogenic, and is largely crucial for muscular tissue growth. The mechanisms and elements responsible for response to ecdysone – a steroid prohormone instigating insect molting events – were also discovered through studies involving the Kc-H line. Since then parallels of the drosophilid ecdysteroid pathway have been discovered in other insects as well, and the roles it plays in the juvenile to adult transition proven. Similar pathways for mammalian metamorphosis have also been described. Knowledge from these findings have additionally been applied to the production of insecticides for pest control purposes.
Though less-defined than S2 or Kc, other drosophilid lines have been used to great effect in addressing conundrums of organism development. For example, introducing fluorescent-tagged marker proteins in assays with such cells has provided a clearer picture where mechanisms underlying cytoskeletal and mitochondrial movement as well as cell-cycle dynamics were previously unknown.
Whilst the variation in the availability of resources for in vivo Drosophila studies is excellent, there is a greater sensibility in undertaking in vitro assays for validation purposes. The quick turnout cell-line based work accommodates means reduced time and costs. Results are also generally more reproducible. As many cell lines remain stable throughout multiple passage cycles, three or more bioreplicates of assays can be executed on cells originating from a singular batch of frozen stock. In studying genetics, the hundred or so well-established cell lines of Drosophila already readily obtainable are diverse enough in nature that one could choose a line which best suits the needs of the experimental design. Due to the simpler population structure of cell lines, immediate observation of cellular response following the addition of a stimulus is also made possible. In our case, plasmids bearing a variety of miRNA-related constructs can be introduced easily, and the effects of overexpression or knockout of such genetic elements characterized within a matter of 48 hours.
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