Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation.
A green fluorescent protein (GFP) cDNA flanked by inverted terminal repeats (ITR) of adeno-associated virus was constructed. The construct sharply improved the efficiency and specificity of the transient expression of genes driven by two general promoters (cytomegalovirus and medaka beta-actin) and one muscle-specific promoter (zebrafish alpha-actin) in transgenic medaka. In addition, treatment with ITR sequence-containing constructs resulted in a dramatic increase in the number of embryos showing uniform GFP-expression at F0. Of the GFP-positive embryos, 34.6% (81/234), 10% (10/60), and 18% (38/212) showed homogenous GFP-expression for the derivative constructs of the cytomegalovirus, alpha-actin, and beta-actin promoters, respectively. As a result of uniform GFP-expression, green fluorescence in founders was (a) extended for an entire lifetime without degradation, and (b) transmitted as a genetic trait to F1 and F2 progeny of some transgenic lines via Mendelian inheritance. A Southern blot analysis revealed a random integration of the transgene into the genome of founders and progeny in both head-to-tail and tail-to-tail concatemerization patterns. Interestingly, some transgenic medaka with uniform and strong fluorescence could be visually noticeable to the unaided eye.
Chou
C Y
CY
Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.
Horng
L S
LS
Tsai
H J
HJ
D000199:Actins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D015139:Blotting, Southern; D003593:Cytoplasm; D004247:DNA / Q000378:metabolism; D005854:Germ Cells / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009990:Oryzias / Q000196:embryology / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D019076:Transgenes
D000199:Actins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA