Zebrafish Conditional Knockout Services

Conditional gene inactivation is necessary for determining the physiological functions of genes whose conventional mutation leads to multiorgan defects or embryonic lethality. It has been widely used in mice due to the availability of embryonic stem (ES) cells and large collections of both ES cells and animals that carry conditional alleles. In the mouse, conditional alleles are commonly generated by introducing two target sites of a site-specific recombinase, either Flp or Cre, in a configuration that will delete an essential portion or an entire gene. In Drosophila, conditional gene inactivation can be achieved by RNA interference (RNAi), and genome-wide libraries of RNAi transgenes are available.

Zebrafish has proven to be a potent vertebrate model for forward genetic analysis of vertebrate development. For the progressively popular zebrafish model, however, only limited conditional alleles generated from invertible gene trapping are available. The lack of ES cells and the low efficiency of RNAi in zebrafish necessitate new methods for targeted conditional gene inactivation. CRISPR/Cas9 technology has been applied to many model systems from cultured cells to whole organisms. Recently, CRISPR/Cas9-mediated integration of loxP sequences for conditional mutagenesis in zebrafish has made its way into the toolkit of zebrafish researchers. In particular, CRISPR/Cas9 mutagenesis coupled with single-stranded oligodeoxynucleotide (ssODN)-mediated repair has simplified experimental design and provided a robust framework for conditional mutagenesis.

CRISPR-Cas9 Mediated Conditional Knockout in Zebrafish

Creative Biogene, a zebrafish research company with experienced scientists and advanced technology, has established a CRISPR-Cas9 mediated method to generate conditional knockout zebrafish models. We can create a floxed allele for tissue- or temporal- specific deletion of your gene of interest. CRISPR/Cas9 system is used to insert loxP sites flanking the region to be deleted in zebrafish. Then, this line can be crossed with a Cre-line expressing the Cre recombinase under a specific promoter or injected with a plasmid containing Cre recombinase. The Cre recombinase promotes recombination of the two loxP sites and the region between the sites is removed from the genome.

Workflow of Zebrafish Conditional Knockout Services

• Fish maintenance and preparation
• Strategy and design
• sgRNA and Cas9 mRNA synthesis
• Microinjection in zebrafish embryos
• PCR and electrophoresis-based genotyping
• Analysis of global gene function and conditionality
• Analysis of tissue-specific function

Applications

• Study the biological functions of certain genes/proteins at the tissue-specific or cellular level
• Building human disease models for gene dysfunction in specific cell types
• Drug screening and development
• Verify cell-type-specific targets drug
• Preclinical safety and toxicity studies

Our conditional knockout services offer the possibility to study gene function in specific cells/tissues. Besides, optimization of these tools allows simultaneous gene inactivation and mutant cell fate analysis by fluorescent cell tracing. Don't hesitate to contact us, we will meet your specific requirements and protect your scientific investment.

References

1. Cornet C, et al. Combining zebrafish and CRISPR/Cas9: toward a more efficient drug discovery pipeline. Frontiers in pharmacology, 2018, 9: 703.
2. Maddison L A, et al. Conditional gene-trap mutagenesis in zebrafish. Gene function analysis. Humana Press, Totowa, NJ, 2014: 393-411.
3. Burg L, et al. Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish. PLoS genetics, 2018, 14(11): e1007754.
4. Yin L, et al. Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs. Genetics, 2015, 200(2): 431-441.

For research use only. Not intended for any clinical use.