Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions

Dongmei Wang, Li-En Jao, Naizhong Zheng, Kyle Dolan, Jessica Ivey, Seth Zonies, Xiaolin Wu, Kangmai Wu, Hongbo Yang, Qingchao Meng, Zuoyan Zhu, Bo Zhang, Shuo Lin, Shawn M. Burgess

Research output: Contribution to journalArticle

84 Scopus citations

Abstract

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F1 off-spring of the infected fish. From F1 fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in ≈50% of the gene "hits," the mRNA transcript levels were reduced by ≥70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F1 fish. This is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F1 fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.

Original languageEnglish (US)
Pages (from-to)12428-12433
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number30
DOIs
StatePublished - Jul 24 2007
Externally publishedYes

Keywords

  • Genetics
  • Retrovirus

ASJC Scopus subject areas

  • Genetics
  • General

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  • Cite this

    Wang, D., Jao, L-E., Zheng, N., Dolan, K., Ivey, J., Zonies, S., Wu, X., Wu, K., Yang, H., Meng, Q., Zhu, Z., Zhang, B., Lin, S., & Burgess, S. M. (2007). Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. Proceedings of the National Academy of Sciences of the United States of America, 104(30), 12428-12433. https://doi.org/10.1073/pnas.0705502104