Transfer of foreign gene to giant freshwater prawn (Macrobrachium rosenbergii) by spermatophore-microinjection.
We developed a spermatophore-microinjection (SMI) technique that allows exogenous DNA fragments to be transferred easily into the giant freshwater prawn (Macrobrachium rosenbergii), an important aquacultural shellfish and aquatic invertebrate model. From 28 to 1, 000 ng of the circular plasmid pGL, in a total volume of 1 microl, were directly microinjected into spermatophores. Fertilization and hatching of prawns created with SMI were completed in vivo. Fertilization and hatching rates in the SMI treatments did not differ from those of the untreated control group. The genomes of free swimming, SMI-created larvae (21 days after fertilization) were analyzed using PCR and Southern blot analyses. A product with a molecular mass of 680 bp was amplified. It corresponded to amplifications of pGL, and Southern blot analysis revealed that the amplified band was positive. The gene transfer rate was primarily dependent on the concentration of DNA during SMI. The higher the concentration of pGL, the higher the rate of gene transfer. PCR and Southern blot analyses detected the existence of foreign DNA in 16 of 23 samples (70%) of genomic DNA isolated from hatched larvae in the 750 ng pGL SMI treatment. SMI, described here for the first time, is the simplest and most efficient method for mass producing transgenic giant freshwater prawns.
Li
S S
SS
Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.
Tsai
H J
HJ
D000818:Animals; D030801:Animals, Genetically Modified; D015139:Blotting, Southern; D005260:Female; D005298:Fertility; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008297:Male; D008845:Microinjections / Q000379:methods*; D033541:Palaemonidae; D016133:Polymerase Chain Reaction; D013093:Spermatogonia / Q000502:physiology*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins