Generation of transgenic Drosophila expressing shRNAs in the miR-1 backbone.

In Drosophila, long-term effects of RNA interference (RNAi) must be achieved by integrating into the genome a template from which an RNAi trigger is transcribed by cellular RNA polymerases, generally RNA polymerase II or III. With encoded triggers, not only can essentially permanent silencing be achieved, but control can also be exerted over the level of trigger expression, with a resulting variation in the degree to which the target is silenced. Knockdown can also be controlled in a temporal and cell-type-dependent fashion through the use of well-established transgenic methodologies and well-tested promoters. The forms of encoded triggers vary. Long double-stranded RNAs can be expressed as extended inverted repeats. The nearest equivalent of a small interfering RNA is an artificial microRNA (miRNA) or short hairpin RNA (shRNA), where a natural miRNA backbone (also called a scaffold) is remodeled to produce a different small RNA or a small inverted repeat (<30 nucleotides) is simply expressed. This protocol describes creation of transgenic Drosophila carrying shRNA inserts in a remodeled endogenous miRNA backbone. The protocol applies to the use of miRNA-based shRNAs, but most of the vectors, principles of experimental design, and methods are also applicable to long inverted repeat transgenes.

Packaging shRNA retroviruses.

To silence a mammalian gene by RNAi using an encoded trigger, a short-hairpin RNA (shRNA) is integrated into the host cell genome as a stable transgene. Target cells are infected with viral plasmid containing shRNA inserted into the vector backbone. Before infection, the plasmid is transfected into a packaging cell line, which provides the trans-acting factors necessary for virus production. These include, minimally, capsid proteins and reverse transcriptase, but they can also include other regulatory factors (e.g., tat for some lentiviral vectors). It is critical to choose the correct packaging cell system for the viral backbone to be used. The packaging cell also defines the host range of the virus, depending on the envelope protein that it expresses. Ecotropic viruses are limited to rodent hosts, whereas amphotropic viruses have a broader host range that also includes humans. Often, investigators will express a nonretroviral envelope, such as vesicular stomatitus virus (VSV) glycoprotein, to enhance virus stability and host range and to enable viruses to be concentrated following production. Although viruses carrying shRNAs are packaged almost identically to viruses carrying protein-encoding genes, one twist is worth noting. shRNAs are efficiently cleaved by the host RNAi biogenesis machinery, which can reduce the level of viral genomic RNAs and consequently viral titers. Therefore, titers can be enhanced by cotransfecting the viral plasmid with a small interfering RNA (siRNA) that targets DGCR-8/Pasha, which is a core microRNA (miRNA) biogenesis component. siRNAs against Drosha can also be used.

Infection of mammalian cells with retroviral shRNAs.

Viral infection is a quite simple approach for stably introducing transgenes (e.g., those encoding short-hairpin RNAs [shRNAs]) into the genome. The critical aspects are that the virus and the target cell should be appropriately matched. For example, a virus bearing an ecotropic envelope protein will not infect a human cell line unless the appropriate receptor has been purposefully expressed. VSV-G (vesicular stomatitus virus glycoprotein) pseudotyped viruses have the greatest host range. Nondividing cells can only be infected with lentiviruses, but the additional safety precautions necessary for the use of these tools should dissuade their application to routinely cultured cell lines.

Creating an miR30-based shRNA vector.

Generating expression constructs for artificial microRNAs (miRNAs) is relatively straightforward. This protocol describes the creation of miR-30-based short hairpin RNA (shRNA) cassettes that are compatible with a number of standard vector systems. The principles outlined here can also be easily applied to other miRNA scaffolds or to simple snapback shRNAs. It is important to note that one must understand the processing of the artificial scaffold and be able to predict precisely the small RNAs that will be generated. Otherwise, no design principles can be effectively applied and the probability that any individual shRNA clone will work effectively will be greatly reduced.

Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells.

The partial purification of mouse mammary gland stem cells (MaSCs) using combinatorial cell surface markers (Lin(-)CD24(+)CD29(h)CD49f(h)) has improved our understanding of their role in normal development and breast tumorigenesis. Despite the significant improvement in MaSC enrichment, there is presently no methodology that adequately isolates pure MaSCs. Seeking new markers of MaSCs, we characterized the stem-like properties and expression signature of label-retaining cells from the mammary gland of mice expressing a controllable H2b-GFP transgene. In this system, the transgene expression can be repressed in a doxycycline-dependent fashion, allowing isolation of slowly dividing cells with retained nuclear GFP signal. Here, we show that H2b-GFP(h) cells reside within the predicted MaSC compartment and display greater mammary reconstitution unit frequency compared with H2b-GFP(neg) MaSCs. According to their transcriptome profile, H2b-GFP(h) MaSCs are enriched for pathways thought to play important roles in adult stem cells. We found Cd1d, a glycoprotein expressed on the surface of antigen-presenting cells, to be highly expressed by H2b-GFP(h) MaSCs, and isolation of Cd1d(+) MaSCs further improved the mammary reconstitution unit enrichment frequency to nearly a single-cell level. Additionally, we functionally characterized a set of MaSC-enriched genes, discovering factors controlling MaSC survival. Collectively, our data provide tools for isolating a more precisely defined population of MaSCs and point to potentially critical factors for MaSC maintenance.

RNAi in cultured mammalian cells using synthetic siRNAs.

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of almost any gene by tapping into innate regulatory mechanisms that are conserved among virtually all eukaryotes. In a typical RNAi experiment, an artificial silencing trigger directs the RNAi pathway toward a target that it would not normally recognize. This is most often an endogenous protein-coding gene, although some noncoding RNAs can also be silenced effectively. The artificial silencing trigger varies; this protocol uses synthetic small interfering RNAs (siRNAs). Lipofectamine 2000 is used to deliver the siRNAs into HEK293 cells. This lipid reagent has proven to be effective for many different cultured mammalian cell lines.