title: Efficient transgenesis mediated by pigmentation rescue in zebrafish.
abstract: The zebrafish represents a revolutionary tool in large-scale genetic and small-molecule screens for gene and drug discovery. Transgenic zebrafish are often utilized in these screens. Many transgenic fish lines are maintained in the heterozygous state due to the lethality associated with homozygosity; thus, their progeny must be sorted to ensure a population expressing the transgene of interest for use in screens. Sorting transgenic embryos under a fluorescence microscope is very labor-intensive and demands fine-tuned motor skills. Here we report an efficient transgenic method of utilizing pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. This was accomplished by co-injecting two constructs with the I-SceI meganuclease enzyme into pigmentless nacre embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation and I-SceI-zpromoter:gene-of-interest-I-SceI to express the gene of interest under a zebrafish promoter (zpromoter). Pigmentation rescue reliably predicted transgene integration. Compared with other transgenic techniques, our approach significantly increases the overall percentage of founders and facilitates accurate naked-eye identification of stable transgenic fish, greatly reducing laborious fluorescence microscope sorting and PCR genotyping. Thus, this approach is ideal for generating transgenic fish for large-scale screens.
authors.affiliation: Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
authors.forename: Itrat
authors.initials: I
authors.lastname: Harrold
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.
authors.forename: Seth
authors.initials: S
authors.lastname: Carbonneau
authors.affiliation: Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
authors.forename: Bethany M
authors.initials: BM
authors.lastname: Moore
authors.affiliation: Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
authors.forename: Gina
authors.initials: G
authors.lastname: Nguyen
authors.affiliation: Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Anderson
authors.affiliation: Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
authors.forename: Amandeep S
authors.initials: AS
authors.lastname: Saini
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.
authors.forename: John P
authors.initials: JP
authors.lastname: Kanki
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.
authors.forename: Cicely A
authors.initials: CA
authors.lastname: Jette
authors.affiliation: Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
authors.forename: Hui
authors.initials: H
authors.lastname: Feng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D018014:Gene Transfer Techniques; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics*; D008856:Microscopy, Fluorescence; D010858:Pigmentation / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015027:Zebrafish / Q000235:genetics / Q000502:physiology; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D051739:Microphthalmia-Associated Transcription Factor; D029961:Zebrafish Proteins; C497237:mitfa protein, zebrafish; D049452:Green Fluorescent Proteins
keywords: pigmentation rescue; transgenesis; zebrafish