Service

Zebrafish knock out service

CRISPR (clusters of regularly interspaced palindromic repeats)/Cas (CRISPR-associated) system is derived from an adaptive immune response against invading phages and plasmids in bacteria and archaea termed type II CRISPR. This genome editing technology is now widely used for making gene knockout/knock-in animals and plants as well as genome-modified cell lines.

TOOLS provides gene-editing services including vector assembly and generating gene knock-out stable clones/Zebrafish/Mice.
Work Flow
1. Provide gene ID or accession number
2. Provide detailed sequence/information if applicable
Step1      Synthesis of CRISPR (guild RNA/recombinant Cas 9 protein) for zebrafish embryos: 3-4 weeks
Step2 Micro-injected CRISPR into embryos: 1 day
Step3 Pre-screen activity/efficiency of CRISPR (extraction of genomic DNA from zebrafish embryos, amplifying sgRNA targeted DNA with PCR, analyze mutation using PerkinElmer LabChip GX Automated high-throughput electrophoresis: 2 days
Step4 Mass scale of microinjection for 120 embryos and then generate F0 founders: 3 months
Step5 Screen germline transmitted F0 founder (analyze mutation by tail fin clipping and select at list 60 somatic mutation F0 fish, out-crossing 30 mutation F0 with wild type fish, identify mutation from embryos using LabChip GX Automated high-throughput electrophoresis, at list 10 germline transmitted F0 founder will be identified: 1-2 weeks
Step6 Select 2 F0 fish from Step5 with ideal mutation and mate F0 with wild type and each one will generate 100 F1 zebrafish: 3 months
Step7 Screen/confirm mutations of 20 F1 by tail fin clipping and accomplish the delivery of 2-4 screened and selectedF1 fish (both sexes but not limited to) to Customer: 1-2 weeks
Step8 Out-cross F1(with mutation) with wild type to generate F2 embryos and accomplish the delivery of F2 embryos to Customer: 1-2 weeks
​Total time : approximately 9 months