Morpholino knockdown and CRISPR/CAS9 genome editing technologies are powerful tools for validating first, and generating later genetic disease zebrafish models. Zebrafish can be used to acquire a deeper understanding on the molecular mechanisms that cause a disease and, importantly, to look for new drugs that might cure or alleviate it.
ZeClinics provides its expertise in transgenic manipulation and zebrafish biology for generating mutant and/or knock-in lines, and later phenotype them, in order to use them for custom drug screenings*.
LICENCED BY THE BROAD INSTITUTE, MIT, HARVARD UNIVERSITY.
*Any of the steps that form this R&D pipeline can be contracted independently. CRISPR/Cas9 Genome-Editing Technology.
Phenotypical validation & drug efficacy assay
|Conditional Knock-out||95%||6 months|
|Conditional Knock-out||100%||1-3 months|
|Targeted gene mutation||100%||1-3 months|
Fact: Large genetic variability that causes disease and limitations of human diseases murine models open the need for innovative models where mutants can be easily validated and generated. Zebrafish allows that directed and high efficient transgenesis, with capability for generating a great number of economic and highly informative disease models. In fact, several genetic mutations with phenotypes that mimic human diseases have been identified, with examples of haematological disorders, solid tumours, heart, muscle, kidney and CNS disorders. Thus, the use of zebrafish models is a very good alternative to existing rodent models of human disease for large scale and low costs drug discovery.
Aim: Generation, validation and use of selected zebrafish disease models for novel drug discovery or other applications.
Assay: The generation of a zebrafish disease model has two phases:
1. Finding and assessing polymorphism/s causing the disease phenotype. We will knockdown the gene of interest by Morpholino injection and co-inject that morpholino with different human versions of the gene: WT and mutated ones. The goal is to revert the phenotype with the WT RNA and to define which are the mutant forms through their incapability to revert the expected phenotype. In addition, this phase will allow us to determine and validate the phenotypes to be reverted in a future screening for drugs to fight the disease. Standard test includes injection of Morpholino plus WT and 4 polymorphic versions of the target gene.
2. Generation of disease model. We will use the previously acquired knowledge to design and generate through CRISPR/Cas9, a novel and powerful genome editing technology, either:
a) Null mutant: We will disrupt the coding frame to generate a complete knock down.
b) Custom mutant: If the goal is to generate a genetic version mimicking a human polymorphism, we will knock-in the gene introducing the desired coding modification.
ZeClinics experience in the use of CRISPR/Cas9 technology is extended to the generation of genetically modified cell lines. Applicable to most of the commercially available human cell lines and induced pluripotent stem cell (iPSC) of your choice. We can design, construct and use the best gRNA to make customized Knockout and knockin lines.
Functional genomic. For a full translational approach select the pack cell + zebrafish and get the clear functional genomic understanding of your gene and/or mutation of interest.
Drug target identification. Customize your iPSC and your patient-derived iPSC with single gene loss of function upon drug lead treatment and select the target of action of your compound.
Just tell us your biological/pharmacological question and we propose and run the best zebrafish-based scientific methodology and provide reliable results. Rare diseases, gene functions, biological pathways, drug-target interaction, drug accumulations and much more can be developed and studied in zebrafish.ASK US ABOUT ZeCUSTOM