Drosophila Conditional KO Service
CRISPR/Cas9 knock-out (KO) has greatly facilitated molecular genetics for destroying gene function specifically, but standard approaches are not suitable for every determination. It is crucial to have the ability to conditionally manipulate the activity of genes, overcoming embryonic lethality of null mutants to study later roles of a given gene, distinguish between cell-autonomous and nonautonomous mechanisms, or to study tissue-specific gene functions. Conditional KO is a worthy and mature strategy in Drosophila causing genetic damage that occurs in a particular time and in a particular tissue or cells.
To manipulate gene expression in a tissue-specific manner via CRISPR, one can utilize two main strategies: (1) restricting Cas9 expression to specific tissues and time or (2) limiting the expression of gRNA to the tissue of interest. In general strategy, tissue specific or conditional inducible promoters are selected to drive the expression of Cas9 protein and sgRNA, so as to achieve gene knockout accurately.
CD BioSciences is a global biotechnology company specializing in specific genomic engineering and analysis in model organism – Drosophila carried out by experienced scientists, advanced laboratory. Partnering with us, you are served with a professional conditional KO Drosophila service that enables you to have rulable control over where or when your gene of interest is knocked out. Relying on rich sgRNA pool and expression systems, we can generate conditional KO Drosophila strains for various time and space knockout conditions.
What is Conditional CRISPR KO?
In order to more precisely regulate the spatio-temporal expression of Cas9 and sgRNA, tissue-specific promoters can be used to drive the expression of Cas9 protein, so as to achieve gene knockout in specific tissues. For example, germline-specific gene knockout can be achieved by using promoters specifically expressed in Drosophila germ line cells to drive Cas9 protein expression. In addition to spatio-temporal regulation of Cas9 expression, spatio-temporal regulation of sgRNA expression can also be achieved by using Pol II promoter with spatio-temporal specificity to drive tRNA-sgRNA expression, and then cutting tRNA to release mature sgRNA through the action of RNA enzymes in vivo. Developing for almost a century, many specific and inducible CRIPSR/Cas9 molecular assembly have been created for different knock out requirements.
The binary Gal4-UAS system is another useful approach to control the expression of Cas9 protein achieving conditional KO Drosophila. In this system, UAS-Cas9 is combined with inducible-Gal4, which drives UAS expression specifically in tissues or under different developing time. Artificial replacement of the Gal4 promotor allows the flies to express Cas9 protein under temporal and spatial control, such as heat shock induced Gal4 (lack the spatial control), light-induced Gal4, or other inducible-Gal4 system strategies. Crossing flies carrying those two transgenes respectively creates tissue-specific gene knock-out progeny with the spatial and temporal control afforded by the Gal4 system.
Since there are quite a lot conditional manipulations in Drosophila conditional KO and each project requires different genes or tissues, we provide every client private conditional KO strategy using specific system and optimal activate way.
Fig.1 Mechanism of Conditional CRISPR Knock-out (KO) using Gal4-UAS System Workflow of Conditional CRISPR KO
We have comprehensive CRISPR/Cas9 editing platform. Either stable genetic strains expressing Cas9 protein and sgRNA respectively or direct injection of Cas9 and sgRNA expression stains can be obtained in our service.
- CONDITIONAL KNOCK OUT DESIGN
- MOLECULAR ASSEMBLY
- DROSOPHILA INJECTIONS
- CANDIDATE SCREENING
- STRAIN CONFIRMATION
We collaborate with you to understand your conditional CRISPR KO expectations. Through our scientific platform, several optimal systems to maximize the chance of successful gene knock -out are designed for clients' choices. We put your requirements and will at the forefront of the project.
We deploy a variety of gene assembly approaches to get the exact sequence needed for appropriate tissue/time-specific expression. We generate efficient the vector or proteins components needed for on-target editing.
Up to 300-embryos are microinjected per sgRNA. Our injection operators have infallible skills and experience. They can obtain precision delivery of transgenesis reagents to Drosophila embryos. We perform a sufficient number of injections that promising >100 crossed G0. And raise the survived larvae to adulthood.
In generally, an exogenous marker or reporter helps 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.
Once we confirm the ends of the gene insertion have proceed as planned, strains homozygous/heterozygote are crossed. Depending on different project, we have G0 and F1 screening by "visible" marker or sequence validation.
G0 or/and F1 are positive screened and confirmed by full sequencing of the entire transgene sequence due to client's opinion. As standard, we validate at the genomic level with advanced sequencing and we provide a functional readout that verifies conditional gene KO efficiency.
During the fruit flies engineering process, our team performs the desired edit(s) defined in the statement of work. We provide regular updates and in-time messages if any issue or complication happen during the manufacturing process.
We ship final transformants in either larvae or adults. Technologically, balancing is necessary if the mutation is homozygous lethal or sterile. We send you F1 homozygous or balanced stable transformants flies. The final transformants vials are maintained in the facility for two weeks as backup.
Application
- Study the biological functions of certain genes/proteins
- Function of non-coding clustered gene families
- Precise analysis of the role of different functional domains
- Build Drosophila mutant library
- Study the important signaling pathways in Drosophila
- Verify targets drug through Drosophila disease model
Why CD BioSciences?
- Record-proven expertise and experience both in genetic engineering and Drosophila.
- Consistent results and superior efficiency for high efficiency gRNA and specific Cas9, achieving ~100% on-target.
- Fast turnaround services to obtain your transformants in less time and lower price.
- Best tracking service with status updates outlining the on-time progress.
CD BioSciences has successfully completed numbers of gene deletion projects through CRISPR/Cas9 platform. We provide you with phased progress and final report of the project implementation. The final report details each step, including genetic transformation strategy, target vector design, construction and verification, genotyping assessment and sequence analysis. We will customize our offering to meet your specific project needs and protect your scientific investment. Since each project is different, if you don't see the CRISPR/Cas9 service you need above, please contact us for more information.
For research use only. Not intended for any clinical use.
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