PUBLICATION
Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish
- Authors
- Huang, C.-J., Tu, C.-T., Hsiao, C.-D., Hsieh, F.-J., and Tsai, H.-J.
- ID
- ZDB-PUB-030902-6
- Date
- 2003
- Source
- Developmental Dynamics : an official publication of the American Association of Anatomists 228(1): 30-40 (Journal)
- Registered Authors
- Tsai, Huai-Jen
- Keywords
- zebrafish, transgene, cmlc2, promoter analysis, heart-specific expression, myocardium, nucleotide mutation, muscle-specific expression
- MeSH Terms
-
- Animals, Genetically Modified
- Zebrafish Proteins/chemistry
- Zebrafish Proteins/genetics*
- Zebrafish Proteins/metabolism
- Exons
- Recombinant Fusion Proteins/metabolism
- Terminal Repeat Sequences
- Myosin Light Chains/chemistry
- Myosin Light Chains/genetics*
- Zebrafish/embryology*
- Zebrafish/genetics
- Cardiac Myosins/chemistry
- Cardiac Myosins/genetics*
- Organ Specificity
- Transgenes*
- Base Sequence
- DNA/chemistry
- Myocardium/cytology
- Myocardium/metabolism
- Gene Expression Regulation, Developmental
- Animals
- Mutation
- Green Fluorescent Proteins
- Time Factors
- Oligonucleotides, Antisense/metabolism
- Luminescent Proteins/metabolism*
- Adenoviridae/genetics
- Promoter Regions, Genetic*
- Germ Cells
- Regulatory Sequences, Nucleic Acid
- Introns
- PubMed
- 12950077 Full text @ Dev. Dyn.
Citation
Huang, C.-J., Tu, C.-T., Hsiao, C.-D., Hsieh, F.-J., and Tsai, H.-J. (2003) Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Developmental Dynamics : an official publication of the American Association of Anatomists. 228(1):30-40.
Abstract
In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (-870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from -210 to 34 (-210/34) was sufficient to drive heart-specific expression, with a -210/-73 motif as a basal promoter and a -210/-174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at -119, evidence that A at -119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping