Biomaterial Database

Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish. 2011

Cong Zhu, and Tom Smith, and Joseph McNulty, and Amy L Rayla, and Abirami Lakshmanan, and Arndt F Siekmann, and Matthew Buffardi, and Xiangdong Meng, and Jimann Shin, and Arun Padmanabhan, and Daniel Cifuentes, and Antonio J Giraldez, and A Thomas Look, and Jonathan A Epstein, and Nathan D Lawson, and Scot A Wolfe

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Zinc-finger nucleases (ZFNs) allow targeted gene inactivation in a wide range of model organisms. However, construction of target-specific ZFNs is technically challenging. Here, we evaluate a straightforward modular assembly-based approach for ZFN construction and gene inactivation in zebrafish. From an archive of 27 different zinc-finger modules, we assembled more than 70 different zinc-finger cassettes and evaluated their specificity using a bacterial one-hybrid assay. In parallel, we constructed ZFNs from these cassettes and tested their ability to induce lesions in zebrafish embryos. We found that the majority of zinc-finger proteins assembled from these modules have favorable specificities and nearly one-third of modular ZFNs generated lesions at their targets in the zebrafish genome. To facilitate the application of ZFNs within the zebrafish community we constructed a public database of sites in the zebrafish genome that can be targeted using this archive. Importantly, we generated new germline mutations in eight different genes, confirming that this is a viable platform for heritable gene inactivation in vertebrates. Characterization of one of these mutants, gata2a, revealed an unexpected role for this transcription factor in vascular development. This work provides a resource to allow targeted germline gene inactivation in zebrafish and highlights the benefit of a definitive reverse genetic strategy to reveal gene function.

UI	MeSH Term	Description	Entries
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D011993	Recombinant Fusion Proteins	Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.	Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015027	Zebrafish	An exotic species of the family CYPRINIDAE, originally from Asia, that has been introduced in North America. Zebrafish is a model organism for drug assay and cancer research.	Brachydanio rerio,Danio rerio,B. rerio,D. rerio,Zebra Fish,Zebra Fishes,Zebra danio,Zebrafishes,D. rerios,Fishes, Zebra,Zebra danios,danio, Zebra
D015252	Deoxyribonucleases, Type II Site-Specific	Enzyme systems containing a single subunit and requiring only magnesium for endonucleolytic activity. The corresponding modification methylases are separate enzymes. The systems recognize specific short DNA sequences and cleave either within, or at a short specific distance from, the recognition sequence to give specific double-stranded fragments with terminal 5'-phosphates. Enzymes from different microorganisms with the same specificity are called isoschizomers. EC 3.1.21.4.	DNA Restriction Enzymes, Type II,DNase, Site-Specific, Type II,Restriction Endonucleases, Type II,Type II Restriction Enzymes,DNase, Site Specific, Type II,Deoxyribonucleases, Type II, Site Specific,Deoxyribonucleases, Type II, Site-Specific,Site-Specific DNase, Type II,Type II Site Specific DNase,Type II Site Specific Deoxyribonucleases,Type II Site-Specific DNase,Type II Site-Specific Deoxyribonucleases,Deoxyribonucleases, Type II Site Specific,Site Specific DNase, Type II
D016335	Zinc Fingers	Motifs in DNA- and RNA-binding proteins whose amino acids are folded into a single structural unit around a zinc atom. In the classic zinc finger, one zinc atom is bound to two cysteines and two histidines. In between the cysteines and histidines are 12 residues which form a DNA binding fingertip. By variations in the composition of the sequences in the fingertip and the number and spacing of tandem repeats of the motif, zinc fingers can form a large number of different sequence specific binding sites.	Zinc Finger DNA-Binding Domains,Zinc Finger Motifs,Finger, Zinc,Fingers, Zinc,Motif, Zinc Finger,Motifs, Zinc Finger,Zinc Finger,Zinc Finger DNA Binding Domains,Zinc Finger Motif
D050989	GATA2 Transcription Factor	An essential GATA transcription factor that is expressed primarily in HEMATOPOIETIC STEM CELLS.	GATA-Binding Protein 2,NFE1B Erythroid Transcription Factor,Nuclear Factor-Erythroid 1B,Transcription Factor GATA-2,GATA Binding Protein 2,GATA-2, Transcription Factor,Nuclear Factor Erythroid 1B,Transcription Factor GATA 2,Transcription Factor, GATA2
D018390	Gene Targeting	The integration of exogenous DNA into the genome of an organism at sites where its expression can be suitably controlled. This integration occurs as a result of homologous recombination.	Gene Targetings,Targeting, Gene,Targetings, Gene