Disruption of Embryonic Development in Common Carp, Cyprinus Carpio, and Channel Catfish, Ictalurus Punctatus, Via Knock Down of BMP2 Gene for Repressible Transgenic Sterilization

Abstract

The primary objective of this research was to evaluate the sterile feral (SF) technology to accomplish reversible transgenic sterilization in fish. Secondary objectives were to 1) determine the ability of SF3 and SF4 gene constructs to interrupt embryonic development, 2) determine the optimum concentration of doxycycline to prevent the interruption of embryonic development, and 3) determine the shortest appropriate time period to apply doxycycline. Transgenic common carp, Cyprinus carpio, and channel catfish, Ictalurus punctatus, embryos were produced from P1 putative transgenic common carp and channel catfish parents that had been electoporated with SF3 or SF4 constructs (modified Tet-off system for knock down of the BMP2 gene). Fifty, 100 or 150 ppm of doxycycline (dox) was applied to some families and replicates of F1 and F2 embryos at different times, 15-20 hours, 20-25 hours, 25-30 hours, 0-30 hours, 30-35 hours, 35-40 hours, 0-60 hours, 30-60 hours after fertilization and 0hr first hatch to rescue embryonic development. There were 2 SF3 families and 4 SF4 families of F1 common carp produced, however, only dox treatment of SF4B3M2 and SF4B4M2 families significantly rescued the transgenic embryos (P < 0.05, ?2). Nine F2 SF3 families and 12 F2 SF4 families were produced by mating F1 male with 2 putative females of each gene construct. No difference in hatchability between dox and no-dox treatments was found (P>0.05 ). Dox treatment of both SF3 and SF4 families significantly increased the hatch and rescued the transgenic channel catfish embryos (P< 0.01, ?2). Dox treatment of SF3 families and Sf4 families which had 0-30, 0-60 and 30-60 treatments increased the hatch and rescued the transgenic embryos ( highest percent was in 30-60 treatment for SF3, and in the 0-30 treatment for SF4) (P< 0.05, ?2). Data from SF3 families suggested that 30-35 treatment increased hatch and rescued transgenic embryos (P<0.01, ?2). The data suggests that the SF approach of embryonic disruption and transgenic sterilization has potential for 100% effectiveness, especially in channel catfish. The F1 and in some cases the F2 generation was reached with promising results for some families. Selection of the correct families and producing the next generation to increase copy number (reaching homozygosity) could make this system completely effective. However, juvenile mortality and decreased growth rate in some families may indicate that the first generation tet-off system may be too leaky and may need to be replaced to obtain optimum results.