INDUCED MUTATIONS IN TICAM 1 GENE OF CHANNEL CATFISH Ictalurus punctatus

Ahmed Elaswad*, Karim Khalil and Rex Dunham
School of Fisheries, Aquaculture and Aquatic Sciences,
Auburn University, Auburn, AL, 36849
*Corresponding author email: ahe0001@auburn.edu  

TIR (Toll / interlukin 1 receptor) domain-containing adapter molecule (TICAM 1) gene has been previously mutated in channel catfish (Ictalurus punctatus) in our lab using transcription activator-like effector nucleases (TALENs) to study the disease resistance to Edwardsiella ictaluri. Upon infection, toll-like receptor (TLR) 3 recognizes pathogen-associated molecular patterns (PAMPs) and recruits and binds its adaptor molecule, TICAM 1, to initiate the signaling pathway. Following E. ictaluri infection, expression of TICAM 1 gene was dramatically down regulated in the spleen and kidney tissues of blue catfish (Ictalurus furcatus), which were more resistant to E. ictaluri than channel catfish (Baoprasertkul et al., Immunogenetics (2006), 58;817-830).

In the current study, a natural columnaris outbreak occurred in one of the tanks holding both mutated and wild-type P1 fish. Mortality continued for 27 days and the number of dead fish was recorded every day. Dead fish were assigned a value representing the day of death while alive fish were assigned 50. A total of 150 (47 mutated and 103 wild-type) fish were used in the study, 89 fish died (27 mutated and 62 wild-type) while 61 survived the outbreak. Genomic DNA was extracted from individual fish, checked for quality and quantity. A 909 bp segment of TICAM 1 gene containing the mutations was amplified with polymerase chain reaction (PCR) using Expand High FidelityPLUS PCR system (Roche).  Mutations were detected using the Surveyor® Mutation Detection Kit (Transgenomic). Surveyor® digestion products were resolved in 2% agarose gel (Figure 1). PCR products were cloned and sequenced to confirm and identify the mutations. Survival curves of mutated and wild-type fish were compared and means of survival time to death was calculated using Kaplan-Meier test.

Mean of survival time to death ± SE was 28.49 ± 1.81 for wild-type fish and 29.64 ± 2.67 for mutated fish.  Percent of fish that survived for the two groups was 42.6% for mutated fish and 39.8% for wild-type fish, however, this was not significant (P>0.05). The fish used in the current study were both mosaic within a tissue as well as heterozygous. Thus, the gene edited fish would be expected to produce TICAM 1, and have the same disease resistance as the wild type fish, which is what was observed.  If knockout of TICAM affects disease resistance, it should be expected in homozygous F1 also when all the cells in all tissues are mutated. The induced mutations in TICAM 1 gene sequence have changed the amino acid sequence however, further investigation is needed to determine if they have any effects on TICAM 1 expression and function.