Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus, by Knockdown of Primordial Germ Cell Genes with Copper-Sensitive Constructs

Abstract

Repressible knockdown approaches were investigated to manipulate for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown and an off-target gene, vasa, was monitored. Two potentially copper-sensitive repressible promoters, yeast ctr3 (M) and ctr3-reduced (Mctr), were coupled with four knockdown strategies separately including: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA), and ds-sh RNA-targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with copper sulfate as the repressor compound. Spawning rates of full-sibling P fish exposed or not exposed to the constructs as treated and untreated embryos were 85 and 54%, respectively, indicating potential sterilization of fish and repression of the constructs. In F fish, mRNA expressions of PGC marker genes for most constructs were downregulated in the untreated group and the knockdown was repressed in the treated group. Gonad development in transgenic, untreated F channel catfish was reduced compared to non-transgenic fish for MctrN2, MN1, MN2, and MDND. For 3-year-old adults, gonad size in the transgenic untreated group was 93.4% smaller than the non-transgenic group for females and 92.3% for males. However, mean body weight of transgenic females (781.8 g) and males (883.8 g) was smaller than of non-transgenic counterparts (984.2 and 1254.3 g) at 3 years of age, a 25.8 and 41.9% difference for females and males, respectively. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but negative pleiotropic effects can result.

Dead-End (dnd) Gene Cloning and Gonad-Specific Expression Pattern in Starry Flounder (Platichthys stellatus)

“…dnd knockdowns can induce sterility in organisms by blocking PGC migration to the gonad developmental region. Genetic engineering techniques have been applied to knock-down dnd in channel catfish ( Ictalurus punctatus ) [ 23 ]. Furthermore, salmon were successfully sterilized by targeting dnd with CRISPR/Cas9 [ 24 ].…”

Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens

“…Repressible primordial germ cell knockout would be one option to accomplish this goal of absolute reproductive control to eliminate virtually all environmental risk of transgenic fish, and this has been accomplished in Channel Catfish (Li et al. 2017, 2018).…”

“…However, for any transgenic approach to be implemented commercially, government regulatory approval will be needed, and that approval is more likely if the fish are reproductively confined. Repressible primordial germ cell knockout would be one option to accomplish this goal of absolute reproductive control to eliminate virtually all environmental risk of transgenic fish, and this has been accomplished in Channel Catfish (Li et al 2017(Li et al , 2018.…”

Comparison of growth performance among channel-blue hybrid catfish, ccGH transgenic channel catfish, and channel catfish in a tank culture system

“…The P 1 transgenic and control broodstock used for this study were from the Catfish Genetics Research Unit, School of Fisheries Aquaculture and Aquatic Sciences, Auburn University, AL, USA. The P 1 transgenic fish were produced via electroporation 63 , 64 . Broodstock were tested to determine if they were transgenic prior to spawning 18 , 27 .…”