Construction And Characterization Of An Activated Nonspecific Cytotoxic Cells (NCC) cDNA Expression Library From Tilapia Peripheral Blood     Kesavannair Praveen, John Leary III, and Liliana Jaso-Friedmann   Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA     The phylogenetic characteristics of the teleost immune system have made fish an important model for the study of vertebrate immunity. We have been analyzing components of innate immunity in fish and have previously reported that NCC are stimulated as measured by increased cytotoxicity and resistance to apoptosis as a consequence of exposure of tilapia to cold temperatures. cDNA  expression libraries are powerful tools to compare  the required molecules necessary for the quiescent and activated states in a specific cell population. An NCC cDNA library was constructed with transcripts from cold-activated tilapia NCC. Total RNA was extracted using Trizol reagent, and mRNA was purified using biotinylated oligo-dT followed by capture with streptavidin immobilized on paramagnetic particles. Purified mRNA was subjected to reverse transcription with StrataScript reverse transcriptase and primed with an oligo(dT) linker containing a Xho I restriction site and 5-methyl dCTP. Double stranded, blunt-end cDNA was ligated with EcoR I adapter to form cohesive ends. Size fractionated cDNA fragments were directionally cloned to the arms of Uni-ZAP XR vector. After in vitro packaging with Gigapack III Gold packaging extract, the XL-Blue MRF’ strain of E. coli was transfected with the lambda phage and recombinants were verified by color selection. The average insert size for the library (as shown by PCR amplification) was 1.1 kb. Ongoing studies are aimed at characterizing the expression of various proteins involved in the activation of innate immune functions mediated by NCC. (Support provided by BARD US 3159-99C).