Efficient expression of transgenes in adult zebrafish by electroporation.
BACKGROUND
Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3-6 month old adult zebrafish.


RESULTS
Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V.cm(-1) at 15 microg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita). To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy.


CONCLUSIONS
Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.
Rambabu
K Murali
KM
Center for Cellular and Molecular Biology, Hyderabad-500 007, India. muralibabu@gmail.com
Rao
S Hari Narayana
SH
Rao
N Madhusudhana
NM
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008156:Luciferases; D009132:Muscles / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D015027:Zebrafish
D049452:Green Fluorescent Proteins; D008156:Luciferases