A large-scale zebrafish gene knockout resource for the genome-wide study of gene function

Thursday, October 11, 2012 — Poster Session III
10:00 a.m. – Noon	Natcher Conference Center, Building 45	NHGRI	GEN/GENOM-26

* FARE Award Winner

Authors

• G.K. Varshney
• J. Lu
• D. Gildea
• H. Hunag
• W. Pei
• Z. Yang
• S. Huang
• D. Schoenfeld
• N.H. Pho
• D. Casero
• T. Hirase
• D. Mosbrook-Davis
• S. Zhang
• L. Jao
• B. Zhang
• T. Wolfsberg
• M. Pellegrini
• S. LIn
• S.M Burgess

Abstract

With the zebrafish genome project completed, it becomes possible to analyze the function of all the zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Currently, there is no high-throughput technology to knockout zebrafish genes in a stable and targeted manner. To address this shortcoming, we developed a streamlined platform using proviral insertions coupled with high-throughput sequencing and mapping technologies to mutagenize zebrafish genome. We developed a highly multiplexed and efficient mapping strategy; this method starts with the use of three sets of restriction enzymes to digest genomic DNA samples in parallel. The digested samples are ligated to 6-base DNA barcode containing linkers and amplicons are generated using linker-mediated PCR. Barcoding allows us to index individual F1 fish, and thus a pool of 500+ fish can be sequenced together. We report the first 5,376 mutagenized and archived F1’s carrying predicted mutations in 3,325 genes. Insertions mapped to the zebrafish genome and mutagenized fish lines are freely available to the public. This resource thus establishes an important milestone for zebrafish genetics research and should greatly facilitate functional studies of the vertebrate genome.

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