title: Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions.
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 F(1) offspring of the infected fish. From F(1) 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 approximately 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 F(1) 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 F(1) fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.
authors.affiliation: Key Laboratory of Cell Proliferation and Differentiation, Center of Developmental Biology and Genetics, College of Life Sciences, Peking University, Ministry of Education, Beijing 100871, China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Wang
authors.forename: Li-En
authors.initials: LE
authors.lastname: Jao
authors.forename: Naizhong
authors.initials: N
authors.lastname: Zheng
authors.forename: Kyle
authors.initials: K
authors.lastname: Dolan
authors.forename: Jessica
authors.initials: J
authors.lastname: Ivey
authors.forename: Seth
authors.initials: S
authors.lastname: Zonies
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wu
authors.forename: Kangmai
authors.initials: K
authors.lastname: Wu
authors.forename: Hongbo
authors.initials: H
authors.lastname: Yang
authors.forename: Qingchao
authors.initials: Q
authors.lastname: Meng
authors.forename: Zuoyan
authors.initials: Z
authors.lastname: Zhu
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Shuo
authors.initials: S
authors.lastname: Lin
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Burgess
mesh_terms: D000818:Animals; D016678:Genome / Q000235:genetics*; D006720:Homozygote; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D020161:Physical Chromosome Mapping; D012333:RNA, Messenger / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D012641:Selection, Genetic; D014158:Transcription, Genetic / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*
chemical_list: D012333:RNA, Messenger