Generación de mutantes mediante tecnología CRISPR/Cas en pez cebra y medaka

Descripción del servicio

This service includes the following steps: 1/ sgRNA design - The customer will provide the location of the genomic region to be mutated. - The customer will receive a PDF with information of the 4 selected sgRNAs. - Approval of the sgRNAs selection is required by the customer in order to proceed with the following step. 2/ sgRNAs transcription - Annealing of the oligos by PCR - sgRNA transcription 3/ Microinjection of the 4 sgRNAs into one-cell stage zebrafish embryos. 4/ F0 injected embryos genotyping to check for in-del mutations. - Genomic DNA extraction from F0 embryos. - Cloning of the region of interest. - Sequencing of the region of interest to check for in-del mutations. 5/ Raising of the F0 injected embryos (4-5 months). - F0 positive embryos are raised to adulthood. - Screening of F0 adult fishes: - Outcross: individual F0 males and females are outcrossed with WT females and males respectively to get the F1 generation. 6/ F1 embryos genotyping. - Genomic DNA extraction from F1 embryos - Cloning of the region of interest - Sequencing of the region of interest to check for in-del mutations. 7/ We provide F1 embryos carrying in-del mutations and a PDF with instructions on F1 adult genotyping. Delivery time: approximately 7 months. Delivery format*: The customer will receive bleached F1 transgenic embryos. * Transport is to be paid by the customer.