2. Expressing Transgenes for the Purpose of Cell Type-Specific Profiling
The vast majority of methods used for cell type-specific profiling require the expression of some sort of transgene in the cells of interest. This is necessary either for sorting/isolating the cells, or to label/pull-down the RNA or DNA from the targeted subpopulation. Transgenes can be expressed through a direct fusion of a promoter to the transgene-coding sequence, or by using a binary system, whereby the promoter is fused to a trans-acting factor, which in turn activates the expression of the effector transgene. In this section we provide an overview of the targeted expression approaches available for each of the common model systems.
2.1. Drosophila GAL4, LexA, and QF Expression Systems
The GAL4/UAS binary system (
Brand & Perrimon, 1993) is the most commonly used method for targeted gene expression in
Drosophila (for reviews, see (
Southall, Elliott, & Brand, 2008;
del Valle Rodríguez, Didiano, & Desplan, 2012)). A wealth of GAL4 “driver” lines, expressing the yeast transcription factor GAL4 in specific cell types, is now available. These “driver” lines can be crossed to specific “responder” lines, which possess upstream activator sequences (UAS) upstream of the transgene to be expressed. In the resulting
Drosophila progeny, the transgene is expressed only in the cells where GAL4 is present (see
Figure 2(A)). Due to the silence of the transgene in the absence of GAL4, responder lines can be generated without the complication of the phenotypic consequences due to misexpression, such as lethality. Additionally, the spatial activity of GAL4 in the organism can be further refined by the use of the GAL4 repressor, GAL80 (
Lee & Luo, 2001;
Ma & Ptashne, 1987). Employing the temperature-sensitive version of the same protein, GAL80ts, enables the temporal selectivity of expression (
Matsumoto, Toh-e, & Oshima, 1978;
McGuire, Le, Osborn, Matsumoto, & Davis, 2003) as well as through the drug-inducible GeneSwitch system (
Osterwalder, Yoon, White, & Keshishian, 2001). Split-GAL4 can also be utilized to produce a more refined expression pattern of the effector, through the intersection of two promoters/enhancers (
Luan, Peabody, Vinson, & White, 2006).
Figure 2 Binary expression systems that can be used for cell-specific profiling.
(A) Binary systems using a transcriptional activator. Shown here is the GAL4/UAS system (
Brand & Perrimon, 1993). One transgenic organism, expressing GAL4 under the control of a specific promoter, is crossed to another possessing UAS sites upstream of a transgene. In the resulting progeny, GAL4 drives expression of the transgene in the cells of interest. (B) The Cre/lox system (
Gu et al., 1993) relies on one parent that expresses the DNA recombinase Cre, crossing to another that has a
loxP-flanked stop cassette between a ubiquitous promoter and the transgene. In the Cre-expressing cells of the progeny, the stop cassette will be removed, allowing the transgene to be expressed.
A recent addition to the
Drosophila tool kit is the Q system, using components identified from the fungus
Neurospora crassa (
Potter, Tasic, Russler, Liang, & Luo, 2010). The Q system is comprised of the transcriptional activator QF, the QF effector QUAS, the QF suppressor QS, and the nontoxic drug quinic acid, which inhibits QS. The Q system can be temporally controlled, when QS is also expressed in the background, through the addition of quinic acid to the food. Recent modifications of the system have produced less toxic versions of QF (
Riabinina et al., 2015). LexA-LexAop from λ phage is a third binary expression system (
Lai & Lee, 2006) which has also been recently updated to utilize the QF activator domain for enhanced expression levels (
Riabinina et al., 2015).
2.2. Mouse Cre/Lox, TetR, and GAL4/UAS Systems
The most common method to drive the expression of a transgene in a cell-specific manner, within mice is the Cre/lox system (
Gu, Zou, & Rajewsky, 1993). There are now a wide variety of transgenic mouse lines that express the site-specific DNA recombinase Cre in specific cell types (
Heffner et al., 2012). Like the GAL4/UAS system, there is a driver line (the mouse line expressing Cre in specific cells) and a responder line (a mouse line that will only express a transgene when Cre is present). However, the method by which the Cre/lox system works is different to that of GAL4/UAS, in that Cre, which is a DNA recombinase, facilitates the removal of a stop cassette positioned between a ubiquitous promoter and the coding sequence of the transgene (see
Figure 2(B)). A stop cassette which comprises a neomycin cassette is often used (
Soriano, 1999). When a Cre-driver line is crossed to a responder line, Cre will bind the
loxP sites flanking the stop cassette and excise it, allowing the ubiquitous promoter to drive expression of the transgene of choice in a permanent and heritable manner. In cells where Cre is absent, the stop cassette is not excised and the promoter is unable to drive expression of the transgene. The Cre/lox system has now been adopted in the vast majority of eukaryotic model organisms due to its success as a site-specific recombinase (
Hubbard, 2014;
Lin, Lee, Wu, Duann, & Chen, 2013;
Vergunst, Jansen, Fransz, de Jong, & Hooykaas, 2000). This form of genetic modification through recombination is very similar to the FLP/FRT system developed in
Drosophila by Golic and Lindquist, which has now also been implemented in mice (
Branda & Dymecki, 2004;
Golic & Lindquist, 1989). For FLP/FRT, the recombinase Flippase (FLP) catalyzes recombination between Flp recognition targets (FRTs) in a setup similar to that described for Cre/lox to induce spatial, and even temporal, transgene expression and knockouts (
Hubbard, 2014).
Choosing an appropriate promoter is an important consideration when designing a responder line. The CAG (chicken beta-actin promoter and cytomegalovirus enhancer) (
Niwa, Yamamura, & Miyazaki, 1991) provides strong expression, especially in neural and heart tissues (
Toyoda et al., 2003). Drawbacks of this promoter, however, include a nonuniformity of expression across tissues (
Griswold, Sajja, Jang, & Behringer, 2011) and the fact that it can be silenced
in vivo (
Rhee et al., 2006). Other widely used ubiquitous promoters are the ROSA26 and UBC (
Kisseberth, Brettingen, Lohse, & Sandgren, 1999;
Schorpp et al., 1996). Homologous recombination can be used to knock-in the transgene directly into the ROSA26 locus and is the preferred option for uniform, ubiquitous expression within the embryo (
Soriano, 1999). More recently, the CAG promoter has been incorporated into the transgene being inserted at the ROSA26 locus (
Madisen et al., 2010;
Snippert et al., 2010). This has been shown to boost expression levels, especially in adult tissues where ROSA26-driven expression is weak (e.g., the brain (
Madisen et al., 2010)).
There are also transcriptional transactivation systems for mice (for a review see (
Lewandoski, 2001)). The tetracycline-responsive system utilizes a tissue-specific expressed TetR-VP16, which only activates transgene expression in the presence of the drug doxycycline (
Gossen et al., 1995). This can be combined with Cre/lox (doxycycline-inducible Cre expression), so that the recombination, and subsequent expression, of a transgene (downstream of a
loxP-flanked stop cassette) can be controlled temporally (
Guo et al., 2005;
Rao & Monks, 2009). Likewise, Imayoshi and colleagues developed a tamoxifen-inducible version of Cre/lox, Nes-CreERT2, which, when crossed with mice harboring ROSA26, enables relative temporal control in the developing nervous system (
Imayoshi, Ohtsuka, Metzger, Chambon, & Kageyama, 2006). Use of site-specific recombinase systems, however, enables the reversible activation or suppression of a transgene, as once recombination event is complete, it cannot be undone. The GAL4/UAS system has also been employed for mouse studies (
Echelard et al., 1993;
Ornitz, Moreadith, & Leder, 1991) and can be temporally regulated by the antiprogestin RU486 (
Wang, DeMayo, Tsai, & O'Malley, 1997).
2.3. Zebrafish GAL4/UAS Expression System
The GAL4/UAS expression system was adapted for use in zebrafish 6
years after its development in
Drosophila (
Brand & Perrimon, 1993;
Scheer & Campos-Ortega, 1999). Since then, the system has been optimized by Distel and colleagues (
Distel, Wullimann, & Köster, 2009). Alterations to the GAL4 driver included the addition of a Kozak sequence, modifications to the codon usage, and insertion of a rabbit β-globin intron to produce the modified GAL4, KalTA4GI. In addition, they optimized the number of UAS sites (x5) and demonstrated that it could be used for permanent labeling of specific cell types through an effector feedback loop.
Temporal control has been recently added to the zebrafish GAL4 tool kit with the development of an inducible system (
Ramezani, Laux, Bravo, Tada, & Feng, 2015). Similar to GeneSwitch in
Drosophila (
Osterwalder et al., 2001), KalTA4 is fused to a mutated ligand-binding domain from the human estrogen receptor, allowing its activity...
PDF (Adobe DRM)
EPUB (Adobe DRM)
