Mei Shang

Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus

Repressible knockdown approaches were investigated 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 salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: 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 sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation.

In vitro digestion of luteinizing hormone releasing hormone analog (LHRHa) using simulated gastric conditions in assessing human food safety.

Luteinizing hormone releasing hormone analog (LHRHa, des Gly10, [D-Ala6] ethylamide) is routinely applied for induced spawning of fish. Simulated gastric acid and pepsin were used to mimic human digestion in vitro to determine the stability of LHRHa upon possible ingestion. In vitro cleavage of LHRHa was quantified using UPLC-MS, ultra-performance liquid chromatography-mass spectrometry. LHRHa was broken down mainly into two fragments from Glp-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (LHRHa) to Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (fragment 1) and Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt (fragment 2). By 24h of incubation, LHRHa was completely digested or barely detectable if the starting material was at 250 ng/μl. If the starting concentration was at 5 ng/μl, LHRHa was completely digested by 5h of incubation, or earlier. In both extreme scenarios, the results indicated that LHRHa would be digested completely in the human stomach and would pose no risk in human food consumption. This study supports the assertion that LHRHa used as a spawning aid would not pose a human food safety risk.