A large-scale insertional mutagenesis screen in zebrafish.
Adam Amsterdam,Shawn M. Burgess,Gregory Golling,Wenbiao Chen,Zhaoxia Sun,Karen Townsend,Sarah Farrington,Maryann Haldi,Nancy Hopkins +8 more
TL;DR: A large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations and cloning a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate.
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Abstract: It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.
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Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.
Yonghua Sun,Bo Zhang,Lingfei Luo,De-Li Shi,Han Wang,Zongbin Cui,Honghui Huang,Ying Cao,Xiaodong Shu,Wenqing Zhang,Jianfeng Zhou,Yun Li,Jiulin Du,Qingshun Zhao,Jun Chen,Hanbing Zhong,Tao P. Zhong,Li Li,Jing-Wei Xiong,Jinrong Peng,Wuhan Xiao,Jian Zhang,Jihua Yao,Zhan Yin,Xianming Mo,Gang Peng,Jun Zhu,Yan Chen,Yong Zhou,Dong Liu,Weijun Pan,Yiyue Zhang,Hua Ruan,Feng Liu,Zuoyan Zhu,Anming Meng +35 more
TL;DR: This work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and the bioinformatics analysis provides some useful guidance to design gene-specific gRNAs for successful gene editing.
Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions
Dongmei Wang,Li-En Jao,Naizhong Zheng,Kyle T. Dolan,Jessica Ivey,Seth Zonies,Xiaolin Wu,Kangmai Wu,Hongbo Yang,Qingchao Meng,Zuoyan Zhu,Bo Zhang,Shuo Lin,Shuo Lin,Shawn M. Burgess +14 more
TL;DR: Using the strategy outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F1 fish, which is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach used in zebrafish for identifying mutations in genes.
Development of a database system for mapping insertional mutations onto the mouse genome with large-scale experimental data.
TL;DR: A database application called MP-PBmice (insertional mutation mapping system of PB Mutagenesis Information Center), developed to serve the on-going large-scale PB insertional mutagenesis project, implemented with the widely used framework Struts-Spring-Hibernate.
A Mutation in Damage-Specific DNA Binding Protein One (ddb-1) Underlies the Phenotype of the No-Marginal-Zone (nmz) Mutant Zebrafish
Kailey Jerome,Aria Gish,Taylor Aakre,Taylor Brend,Christina L. Johnson,Jaxon Gronneberg,Lucas Radermacher,Tristan Darland +7 more
Abstract: The ciliary marginal zone (CMZ) is a region in the peripheral-most retina that displays ongoing retinogenesis during growth and expansion of the eye in adulthood. While there is evidence that this capacity also exists in birds and mammals, it is far more robust in fish and amphibians. The process of CMZ retinogenesis is essentially equivalent to that seen early in the central retina; however, its regulation is not fully understood. In a previous study, we attempted to uncover novel regulatory genes by using a forward genetics screen in zebrafish, looking for recessive CMZ mutants. One of the mutants found was called no marginal zone (nmz). The nmz mutant showed relatively normal central retina development, but a lack of cells in the CMZ by 5 days post fertilization (dpf). Mapping, genomic sequencing, and complementation analysis using a second mutant line (m863) isolated in another laboratory showed that a mutation in damage-specific DNA binding protein-1 (ddb-1) gene underlies the phenotype seen in nmz. BrdU labeling suggested that later expansion and differentiation of CMZ retinal progenitors is more affected by ddb-1 loss than the earlier process of stem cell asymmetric division. As was seen for the m863 mutant and in other studies with mice, one profound effect of ddb-1 loss in nmz was the upregulation in expression of tp53 and several of its downstream effectors. Several important genes important in CMZ retinogenesis are also downregulated in the nmz mutant. The change in gene expression would suggest that ddb-1 loss leads to increased cell cycle disruption and apoptosis at the expense of CMZ retinogenesis. While homozygosity is lethal, heterozygous fish appear to be completely normal in morphology, visual function, and behavior.
The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells
TL;DR: Frog Prince is the most efficient DNA-based transposon from vertebrates described to date, and shows approximately 70% higher activity in zebrafish cells than SB, and can greatly extend the possibilities for genetic analyses in vertebrates.
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