Academic journal article Genetics

Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase

Academic journal article Genetics

Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase

Article excerpt

THE ability to introduce exogenous DNA sequences (transgenes) into the genome of an organism or cell line (transgenesis) underpins much of modern molecular genetics and experimental biology. Transgenes are used to label cells, to manipulate physiology and behavior, and to test hypotheses about gene function. Hence, it is essential that the DNA sequences carried within a transgene be expressed at functional levels and with high spatial and temporal specificity. Yet endogenous regulatory elements frequently exert a strong influence over the expression of a locally inserted transgene, and it is common for two insertions of the same transgene to behave differently due to their distinct local chromatin environments (Lewis 1950; Spradling and Rubin 1983; Levis et al. 1985; Akhtar et al. 2013). Such position effects (PE) may lead to spatial and temporal mis-regulation, overexpression, or silencing of transgenes, which can seriously confound the interpretation of experimental results.

Transgenesis strategies that allow researchers to control the genomic position of transgenes present an opportunity to standardize PE across transgenes and experiments. Among these, site-specific transgene integration, mediated by a bacteriophage-derived integrase, is an especially popular technique due to its high rate of transgenesis and ability to function in a broad range of species (Smith et al. 2010; Geisinger and Calos 2013). The integrase from phiC31 has been deployed in multiple eukaryotes, including fruit flies (Groth et al. 2004), zebrafish (Mosimann et al. 2013), tobacco (Lutz et al. 2004), and cultured human cells (Groth et al. 2000). Three components are required to integrate a transgene. The first component, the integrase protein (Int), mediates recombination between two specific DNA sequences called attP and attB (Kuhstoss and Rao 1991). The second component is the transgene to be integrated, which carries the attB sequence. The third component consists of a stable, molecularly mapped transposon insertion that carries the attP sequence; this genomic "landing site" serves as the platform into which the transgene is integrated. This organization gives Int-mediated transgenesis incredible flexibility: any transgene with attB can be integrated, and the genomic position of a transgene can be controlled based on landing site selection. Yet despite its considerable advantages, Int-mediated transgenesis is constrained in many systems by a lack of well-characterized landing sites.

The primary measure of landing site quality is whether integrated transgenes express faithfully and at experimentally relevant levels. In the fruit fly Drosophila melanogaster,anumber of groups have constructed collections of landing sites (Groth et al. 2004; Venken et al. 2006; Bischof et al. 2007; Markstein et al. 2008; Ni et al. 2009), and two studies have explored the prevalence of PE by comparing the expression of reporter transgenes integrated at multiple landing sites across thegenome(Marksteinet al. 2008; Pfeiffer et al. 2010). Using a Gal4-inducible luciferase transgene integrated at 20 different landing sites, Markstein et al. (2008) observed both variable Gal4-inducedexpressionand"leaky" expression of luciferase in the absence of Gal4. Furthermore, for a given landing site, the authors observed that the degree of luciferase induction in one tissue or cell type was not predictive of expression in other tissues or cell types (Markstein et al. 2008). Given that many landing sites (perhaps most) are subject to undesirable PE (Markstein et al. 2008; Pfeiffer et al. 2010), it is clear that landing sites should be tested for deleterious PE in the cell types or developmental stages of interest before embarking on an experiment. However, assessing large numbers of landing sites is time- and resource-intensive (Markstein et al. 2008; Pfeiffer et al. 2010) and imposes a substantial burden on the research community. Thus, a strategy that streamlines the screening of landing site candidates would expedite the generation of transgenic resources tailored to specificexperimental needs. …

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