Transfer of a foreign gene to Japanese abalone (Haliotis diversicolor supertexta) by direct testis-injection

We developed a technique that easily transfers exogenous DNA fragments into the genome of Japanese abalone (Haliotis diversicolor supertexta), an important aquaculture shellfish. From 0.1 to 10 μg/μl of the linearized plasmid pOBA–YPGHc, containing the yellowfin porgy (Acanthopagrus latus) growth hormone cDNA driven by the medaka β-actin promoter, was directly injected into abalone testis with a microsyringe in a total volume of 10 μl. The sperm activity, fertility, and hatchability of fertilized eggs from testis-injected abalone did not differ (P < 0.05) from those of abalone in the untreated control group. From extracted genomic DNA from sperm, larvae at the trochophore stage (9 h after fertilization), juveniles (3 weeks after fertilization), and adults (1-year-old) of testis-injected abalone, a product with a molecular weight of 954-bp was amplified using polymerase chain reaction (PCR). Southern blot analysis showed that the amplified PCR product hybridized to the pOBA–YPGHc probe. The gene-transfer efficiency of G0 in larvae, juveniles, and 1-year-old adults was 90%, 92.5%, and 60%, respectively. Around 20% of G0 mature abalone contained the transgene in their gonads. Genomic Southern blot analysis of 1-year-old abalone derived from DNA-injected group revealed that the transgene was integrated in the genome of the transgenic abalone. The mean shell lengths and body weights were significantly greater (P < 0.05) than those of control group. We conclude that direct testis injection is a simple, high-throughput, minimally invasive, non-viral based, and efficient approach for producing transgenic abalone.