From Classic Genetics to Cutting-Edge Biotech: Exploring the Genetic Tools Enabling Protein Expression in Fruit Flies

Contributors: Sophie Keegan PhD, Connor Davis BSc, Anzer Khan PhD, Diane Jeon BComm, Simon Wu MSc

In biotechnology today, there is a growing demand for recombinant proteins across multiple sectors, and specifically a demand for manufacturing capacity and capabilities in producing difficult-to-express proteins (DTEPs). Protein expression is a complex activity–from transcription to translation with hundreds of interrelated variables at play. The interplay between these variables becomes increasingly important for proteins involved in disease treatment, cell therapies, and the like.  

Current expression systems are not adequately equipped to produce these more intricate protein targets. To meet this growing demand, there is a need for novel expression systems that are both flexible and scalable. The EntoEngine™ system leverages the genetically amenable Drosophila melanogaster as an expression host to tackle the increasing need for complex proteins.  

Drosophila melanogaster has served as a vital model organism for research for over a century. Generations of scientists have established an extensive array of tools that make Drosophila an invaluable tool for research. At Future Fields, we build on this century-long history to advance Drosophila into the arena of recombinant protein production and bridge the gaps in technical capabilities of existing systems. 

This article will serve as a valuable resource for those seeking to understand the genetic capabilities available to express complex proteins. We will highlight the unique genetic characteristics of Drosophila as an expression host, and how we are utilizing this organism to overcome challenges faced in the production of recombinant proteins.

But first, an EntoEngine™ process overview

Before exploring the genetic advantages of transgenic insect expression systems, it is important to understand how genetics fits into our process for recombinant protein production. Our process is summarized in the video below.

The genetics portion of our process is made up of three main steps:

1. Integrating our gene of interest into the genome of Drosophila.

2. Optimizing recombinant protein expression using genetic tools in Drosophila.

3. Establishing and monitoring our stable populations of flies as manufacturing is scaled.

These three stages offer unique advantages of using Drosophila melanogaster for protein production. We will dive into each step to highlight the benefits for those seeking alternative platforms with more genetic capabilities.

1. Stable incorporation: Integrating our gene of interest into the genome of Drosophila

The protein expression process starts with the genetic sequence. In order to produce a recombinant protein in Drosophila, the genetic sequence encoding for that protein needs to be introduced into the Drosophila cells. To do this, we stably incorporate a DNA sequence from humans or other species into the genome of Drosophila.

Stable integration of a genetic sequence in Drosophila is a simple and cost-effective technique which is performed by injecting a plasmid into Drosophila embryos (Rubin and Spradling, 1982). This plasmid includes three important elements: 1) a sequence that will allow integration into the genome, 2) the gene encoding the protein of interest, and 3) a gene which will produce a phenotype (e.g. eye color) to track successful integration. Using this method, hundreds of injections can be performed for a single plasmid and successful integration can be detected by a simple visual check for a change in eye color. Once transformed flies are identified, they are propagated to expand the population while maintaining the inserted gene in subsequent generations.

Once the gene has been incorporated into the genome, the desired protein can then be expressed. Despite their apparent lack of similarity to humans, Drosophila are well suited for the expression of human proteins because they exhibit strong evolutionary conservation of genes and molecular mechanisms. This is perhaps best exemplified by the finding that ~70% of genes that are linked to disease in humans have orthologs (i.e. genes evolved from a common ancestor) in Drosophila (Rubin et al., 2000, Reiter et al., 2001, and Yamamoto et al., 2014). This genetic similarity between humans and fruit flies gives Drosophila an advantage over unicellular model organisms like bacteria, offering more capabilities for human protein expression. For challenging protein targets, we are able to utilize genetics tools to gain access to these capabilities offered by Drosophila to optimize protein expression.

2. A tunable expression system: Optimizing recombinant protein expression using genetic tools in Drosophila.

With the increased demand for DTEPs, there is a need for more diverse expression systems. A major benefit of using Drosophila as an expression system is that they contain multiple different cell types within a single system. They are also living organisms that progress through developmental stages. This means that there is more flexibility to express a protein in a specialized cell type or at a specific developmental stage, which is not possible in cultured cell expression systems that use a single cell type in a stable state. We are able to tap into these diverse cell types and stages by using genetic tools to target gene expression.

Drosophila as an expression system is remarkably tractable, with one of the most extensively used tools being the GAL4-UAS system (Brand and Perrimon, 1993). This system, which was adapted from yeast, allows for gene expression to be turned on in specific tissue types in Drosophila (tissue-specific expression). GAL4 is a transcriptional activator that can bind to the upstream activating sequence (UAS) to drive gene expression. An extensive catalog of fly lines have been developed that express GAL4 in different tissue types (see FlyBase), which can be mated to others that have a UAS next to our gene of interest. This method allows the generation of flies that only express our protein of interest in a specific tissue. Targeted expression is especially powerful for very elaborate proteins that may require expression in specialized cells that contain the necessary components to make that protein functional (Feizi et al., 2017, Labbadia and Morimoto, 2015). Tissue specific expression can be optimized for processes such as protein folding, oligomerization, glycosylation, and other post-translational modifications (PTMs) required for full functionality. For proteins that do not require a tissue-specific approach, a ubiquitous promoter can be used to express our protein of interest in all tissue types, ensuring maximum protein yield.

As an alternative method to control protein expression, our gene of interest can be inserted into the Drosophila genome with an inducible promoter. Inducible expression is a key feature of our system to prevent gradual loss of protein expression over time, which is a significant challenge commonly faced in the recombinant protein production industry (Barnes et al., 2003, Kim et al., 2011). Loss of expression in other systems is primarily due to genetic instability or the metabolic burden placed on the cells from continuous protein over-expression (Misaghi et al., 2014, Dahodwala and Lee, 2019). By contrast, our inducible system maintains the integrity of gene expression by avoiding prolonged, unnecessary protein production. This not only enhances the long-term stability and health of our flies, but also allows us to optimize conditions for each target protein. We make use of an inducible system with a temperature sensitive promoter which is activated simply by shifting the flies to higher temperatures, making this a cost-efficient method for inducible protein expression.

When combined, the ability to express proteins in many different tissue types, genetic tools like the GAL4-UAS system, and the use of inducible promoters make up a protein expression platform that is both versatile and customizable (Figure 1). This flexibility of Drosophila as an expression system allows for the expression of even the trickiest protein targets with the EntoEngine™.

Figure 1: Animation demonstrating how the GAL4-UAS system functions, including various methods of inducing the GAL4 system.

3. Easy maintenance and traceability of new fly lines

Once a new fly line is generated and protein expression optimized, the maintenance of this line is one of the most significant advantages to our system compared to traditional protein expression systems. To make recombinant proteins in mammalian cell cultures, cells are grown in bioreactors, requiring precise growth conditions and costly cell media (Dahodwala and Lee, 2019). These cultures are also prone to instability and loss of expression, which is not easily detectable. Drosophila are able to overcome both of these challenges, without the need for specialized equipment or bioreactors.

Drosophila are easy to maintain in a laboratory setting compared to other expression systems. A major advantage is the simplicity of rearing—standard fly food, made from simple household ingredients provides all the necessary nutrients for their growth. Neither antibiotics or expensive growth media are required in the process. Drosophila stocks are typically kept at two different temperatures. Long-term stocks are maintained at 18°C, where flies develop more slowly, reducing the need for frequent handling. These stocks are usually transferred to fresh food once a month. On the other hand, genetic crosses, routine lab work, and scaling are performed at 25°C, where the life cycle of the flies is much faster, taking about 10–12 days from egg to adult. Rearing flies at 25°C means populations can be scaled quickly, in turn producing more recombinant protein in a shorter time frame.  

Throughout the scaling process, we are able to monitor the status of our fly stocks using easily traced visual markers. As described above, a visual marker is always inserted with our gene of interest to confirm successful integration. This visual marker is also used to monitor the long-term stability of the gene of interest in our stocks and to generate new expression stocks. For example, to change the expression strategy for a particular protein, flies can simply be mated to a different GAL4 line, generating a new stable stock in a few weeks thanks to visual markers. This process is faster than genotyping, as the visual markers provide immediate, observable phenotypes, eliminating the need for labor-intensive molecular testing.

Some common visual markers such as eye color or shape, body color, and wing shape are highlighted in Figure 2. These visual markers are also often associated with balancer chromosomes, as will be discussed below.

Figure 2: Phenotypic markers help track the inheritance of specific alleles or chromosomes across generations, enabling the identification of fruit flies that are expressing the target protein.

While visual markers inserted with our gene allow us to detect loss of that gene, we want to minimize the probability of this occurring, especially when fly populations are grown at scale. The key genetic tool that allows us to maintain stable expression stocks essentially in perpetuity is the balancer chromosome. Balancer chromosomes have two key functions: first, they allow the population of flies to be maintained as heterozygotes, and second, they suppress meiotic recombination. To address the first point, maintaining heterozygous populations is beneficial because flies with two copies of a transgene (homozygotes) may not be viable if the inserted transgene has harmful effects on the health of the fly. In cases where the inserted gene is homozygous lethal, balancer chromosomes ensure that progeny always inherit one chromosome bearing the transgene and one balancer chromosome. Balancer chromosomes also contain recessive lethal mutations which prevent progeny from becoming homozygous for the balancer.

The other essential function of balancer chromosomes is to suppress recombination, which is the exchange of genetic material between sister chromatids (Hunter, 2015). In natural populations, recombination is important for generating genetic diversity. When developing transgenic fly lines however, recombination can be problematic, leading to loss of the desired transgene from a portion of the progeny. Balancer chromosomes contain multiple inversion breakpoints that disrupt homologous recombination during meiosis, ensuring that the gene of interest is not lost (Ashburner et al., 2005). Balancer chromosomes allow our fly stocks to stably maintain a single copy of an inserted transgene over many generations.

In addition to the above mentioned features, balancer chromosomes also carry dominant markers, such as curly wings, which simplify the tracking of gene inheritance during genetic crosses without requiring genotyping. This enables straightforward selection for or against specific phenotypes, making it easier to identify flies with the desired genotype. The use of balancers in our process is visualized in the figure below (Figure 3). These features make balancer chromosomes an invaluable tool in genetics and our scale up process, helping to maintain stable lines of flies with desired genetic modifications.

Figure 3: A genetic cross with possible gene and balancer combinations in the progeny. Homozygosity for the balancer is lethal, so the gene is maintained in a heterozygous or homozygous state within the population.

The simplicity in rearing Drosophila, along with the use of visual markers and genetic tools such as balancers, allow us to scale efficiently and reduce costs. We pass these cost benefits on to our custom protein clients.

Celebrating the Drosophila melanogaster research community

At the heart of the EntoEngine™ lies the rich heritage of Drosophila genetics, built on over a century of research. With the solid foundation created by this research community, we are able to offer a cutting-edge, customizable solution that can tackle the challenges of protein production.

Several notable resources from the Drosophila research community include FlyBase, The Bloomington Drosophila Stock Centre, and The Drosophila Genomic Resource Centre. Thanks to the wide range of tools available, Drosophila offers unmatched capabilities that can meet your needs today, whether you are looking to scale up production or express complex, human-like proteins.

Curious about how you could leverage the genetic advantages of the EntoEngine™ for your protein research and production goals? Reach out below.

Sources

1. Ashburner, M., Golic, K. G., & Hawley, R. S. (2004). Drosophila: a laboratory handbook.
2. Barnes, L. M., Bentley, C. M., & Dickson, A. J. (2003). Stability of protein production from recombinant mammalian cells. Biotechnology and Bioengineering, 81(6), 631-639.
3. Brand, A. H., & Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, 118(2), 401-415.
4. Dahodwala, H., & Lee, K. H. (2019). The fickle CHO: a review of the causes, implications, and potential alleviation of the CHO cell line instability problem. Current Opinion in Biotechnology, 60, 128-137.
5. Feizi, A., Gatto, F., Uhlen, M., & Nielsen, J. (2017). Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome. NPJ systems biology and applications, 3(1), 22.
6. Hunter, N. (2015). Meiotic recombination: the essence of heredity. Cold Spring Harbor Perspectives in Biology, 7(12), a016618.
7. Kim, M., O'Callaghan, P. M., Droms, K. A., & James, D. C. (2011). A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnology and Bioengineering, 108(10), 2434-2446.
8. Labbadia, J., & Morimoto, R. I. (2015). The biology of proteostasis in aging and disease. Annual Review of Biochemistry, 84(1), 435-464.
9. Misaghi, S., Chang, J., & Snedecor, B. (2014). It's time to regulate: Coping with product‐induced nongenetic clonal instability in CHO cell lines via regulated protein expression. Biotechnology Progress, 30(6), 1432-1440.
10. Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., & Bier, E. (2001). A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Research, 11(6), 1114-1125.
11. Rubin, G. M., & Spradling, A. C. (1982). Genetic transformation of Drosophila with transposable element vectors. Science, 218(4570), 348-353.
12. Rubin, G. M., Yandell, M. D., Wortman, J. R., Gabor, G. L., Miklos, Nelson, C. R., ... & Lewis, S. (2000). Comparative genomics of the eukaryotes. Science, 287(5461), 2204-2215.
13. Yamamoto, S., Jaiswal, M., Charng, W. L., Gambin, T., Karaca, E., Mirzaa, G., ... & Bellen, H. J. (2014). A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases. Cell, 159(1), 200-214.