Gal4 RNAi in Drosophila

In Drosophila, RNAi is achieved by expressing artificial inverted repeat (IR) sequences complementary to related genes. Gal4 inducible RNAi system is attained when UAS is put in front of the IRs. IR has a natural hairpin structure, so it is also called RNAi hairpin. According to the length of IR sequence, it is divided into long hairpins and short hairpins RNA (shmiR). The long IRs size ranges between 109 and 600 bp. And shmiR oligo has around 21 nt cloned into artificial hairpins to perform RNAi, increasing the specific of gene knock-down. When UAS-IR is activated by Gal4 in cells, IR will be expressed. And long hairpins will be cut into functional small interfering RNAs (siRNAs). Then siRNAs or shmiR are loaded into RISC. And the activated complex elicits gene silencing by binding, through perfect complementarity, to target mRNAs, thereby targeting it for cleavage and degradation.

Fig.1 Gal4 RNAi 

• GAL4 RNAi STRATEGY

We provide you with the most convenient and effective strategy for your research purpose. Full communication and years of experience allow us to make a comprehensive plan, detailing selection of optimal RNAi target sites, optimizing Drosophila codon sequences, adding selectable marker, assembly of appropriate UAS-RNAi complex, selection of specific Gal4 lines, production of stable transgenic fruit flies to maximize the chance of success. Moreover, we provide other one-stop services for further gene function or interaction researches.

• CONSTRUCTS

Depending on the project approach, we offer optimal UAS-IR vectors and composite Gal4 lines generation. With our UAS-RNAi library, we provide transgenic vectors ranging from somatic to germ cells, from tissues to developing time, or at different knock-down intensity. Common selectable markers are needed for quick screening, like vermillion, mini-white, etc. 3 specific hairpins to different regions are designed per target gene. In addition, we improve the efficiency of RNAi by targeting serval types mRNA of the same gene. We have systematic strategy to minimize the chance of off-target effects, especially in conducts design process.

• BALANCING

Once the injection is down, the survived larvae will be raised to adulthood. Unless specifically requested, we inject wild-type fruit flies. We provide homozygous. But technologically, balancing is necessary if the mutation is homozygous lethal or sterile.

• VALIDATION

In generally, visible markers help identify candidates. But there are no limits in screening pathways. For our genomic integrant, we look at each transgenesis (including effector and transactivator transformants) by PCR.

A series of genetic crosses to remove the integrase source, to establish a homozygous stock and to construct stable lines that carry Gal4 driver compose and UAS-IR. As standard, we validate at the genomic level with advanced sequencing and we provide a functional readout that verifies system efficiency and flies' vitality.

• PHENOTYPIC ANALYSIS 

We analyze RNAi-mediated gene knock-down leading to one or more phenotypes in Drosophila to determine the effect of RNAi. The effect of RNAi is evaluated using various techniques to quantify RNA or from the protein level (or by omics) due to clients' need. Furthermore, knock-down results can also be determined by visualizing protein immunofluorescence (optimal).

We provide regular updates and in-time messages if any issue or complication happen during the manufacturing process. The final report details every manufacturing process and analysis data will feed back to you with the flies.

We ship final transformants in either larvae or adults. The final transformants vials are maintained in the facility for two weeks as backup.

At CD BioSciences, we offer complete services to improve and extend clients' researches in Drosophila. We pioneer advanced approaches to assist customers around the world access high-quality solutions and supports.

• With our UAS-RNAi library, we provide RNAi services to repressing genes ranging from somatic to germ cells, for example, mosaic analysis with a repressible cell marker (MARCM).
• We provide other customized gene knock-down services. RNAi experiments can be combined with additional Gal4-UAS tools, Gal80^TS, GeneSwitch and Split Gal4, etc., to restrict knockdown to specific spatial-temporal windows, or at different knock-down intensity.
• Further connection services, including sample preparation, immunofluorescence imaging analysis and other qualitative and quantitative phenotypic analysis services.
• High throughput RNAi screening.
• Establishment of shRNA and UAS-RNAi vector libraries.

• Identification novel genes and pathways
• Individual functions and contributions to cellular phenotypes
• Cell differentiation and proliferation of germline
• Validating targets for diseases including cancer and genetic diseases