Molecular and Immunologic Characterization of Gynogenetic Channel Catfish (Ictalurus punctatus)

Abstract

: Second-generation gynogenetic channel catfish were characterized by molecular and immunologic assays to determine if they were isogenic at major histocompatibility complex loci. Southern blot analyses, using channel catfish MHC class II B and class I A gene probes, revealed identical banding patterns among second-generation gynogenetic fish. In contrast, banding patterns from outbred fish differed not only from gynogenetic animals, but also among themselves. Nucleotide sequence analysis of the MHC class II beta(1) domain, which encompasses the peptide binding region, from four randomly selected gynogenetic fish showed a single DNA sequence. In contrast, analysis of the same region from three outbred fish showed sequences that differed not only among themselves, but also from those of gynogenetic animals. In cytotoxic assays, peripheral blood leukocytes from outbred fish lysed both gynogenetic and allogeneic targets, whereas those from gynogenetic fish lysed only allogeneic targets. Taken together, these results suggest that this group of second-generation gynogenetic channel catfish is isogenic at MHC loci and may provide an excellent system with which to study cell-mediated immunity in teleosts.

TheIgHLocus of the Channel Catfish,Ictalurus punctatus, Contains Multiple Constant Region Gene Sequences: Different Genes Encode Heavy Chains of Membrane and Secreted IgD

“…Outbred channel catfish (1-2 kg) were obtained from ConAgra (Isola, MS) and second-generation homozygous MHC-matched gynogenetic channel catfish were obtained from the Catfish Genetics Research Unit, U.S. Department of Agriculture-Agricultural Research Service (Stoneville, MS) (16,17) and maintained in individual tanks (18).…”

A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus

“…To perform immunizations, gynogenetic catfish were anesthetized with tricaine methanesulfonate (MS-222; Sigma-Aldrich, St. Louis, MO) and immunized intracoelomically with 3 3 10 6 G14D cells that were infected 3 h earlier with CCV at a multiplicity of infection of 10 (G14D-CCV) or injected with 3 3 10 6 mock-infected G14D. G14D is a clonal T cell line that is MHC-matched to our gynogenetic fish (40). The Auburn CCV strain was propagated in channel catfish ovary cells as described previously (48).…”

“…The 3B11 cell line is a cloned autonomous B cell line generated from an outbred catfish by mitogen stimulation (50), 42TA is a macrophage cell line that contains some T cells (51), 28S.3 and G14D are clonal autonomous T cell lines (40,52), and TS32.15 is a cloned nonautonomous Ag-dependent CTL, which requires weekly stimulation with irradiated catfish 3B11 B cells for continuous proliferation (28). TS32.15 cells were grown in AL-5 medium containing 5% each of 42TA and 28S.3 culture supernatants.…”

“…In this study, we use our model of homozygous gynogenetic fish and the MHC-matched G14D clonal T cell line to study catfish antiviral cytotoxic cell responses (40). Importantly, G14D T cells support the replication of channel catfish virus (CCV), also known as Ictalurid herpesvirus-1.…”

B cell receptor accessory molecules in the channel catfish, Ictalurus punctatus

“…Genomic DNA was prepared using erythrocytes from outbred and homozygous gynogenetic [49] catfish as previously described [50]. Briefly, genomic DNA (10μg) was digested to completion with EcoR I, or Pst I restriction enzyme, separated on 1% agarose gels and transferred by capillary action onto Hybond-N+ membranes (GE Healthcare Bio-Sciences AB, Piscataway, NJ) using standard techniques.…”