Electroporation of adult zebrafish.
We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3-6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 mug/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1alpha, and Xenopus EF-1alpha) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism.
Rao
N Madhusudhana
NM
Center for Cellular and Molecular Biology, Hyderabad, India.
Rambabu
K Murali
KM
Rao
S Harinarayana
SH
D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D003532:Cyprinodontiformes; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases