Construction of transgenic P₀ grass carp by capsid-targeted viral inactivation of reovirus

To explore new methods to prevent the grass carp(Ctenopharyngodon idella)hemorrhage,the capsid-targeted viral inactivation(CTVI)strategy was developed in the present study.By combination with Cyprinus carpio heat shock protein 70 or Xenopus EF1α promoters,two different transgenic plasmids pTgf2-EF1α-VP3-SN and pTgf2-Hsp70-VP3-SN were constructed harboring with efficient Tgf2 transposon element.Both transgenic plasmids contain open reading frame(ORF)fusion with GCRV capsid protein VP3 and Staphylococcus aureus nuclease(SN).The transgenic grass carp was produced by co-injection pTgf2-EF1α-VP3-SN or pTgf2-Hsp70P-VP3-SN plasmids into 1-2 cell fertilized eggs with in vitro synthesized Tgf2 transposase mRNA.The PCR and sequencing analysis have proved that the anti-GCRV transgenic systems have successfully been integrated into the genome of grass carp.The transgene positive rates of pTgf2-EF1α-VP3-SN or pTgf2-Hsp70P-VP3-SN plasmids are 40.2% and 37.0%,respectively.Our results demonstrated Tgf2 transposon can efficiently mediate transgenesis in grass carp for fusion ORF of GCRV capsid protein VP3 and SN.Total 120 transgenic grass carp individuals have been obtained in the present study.The construction of anti-GCRV transgenic P₀ will provide the materials for future transgenic breeding to prevent grass carp hemorrhage.