70 Present address: 1 Research Associate (uttaraivri@gmail.com), 3 Senior Research Fellow (lakshyaveer@gmail.com), 4 Scientist (rush2aditya@gmail.com), 5 T-9 (sudeshivri@gmail.com), 7 Senior Scientist (gandham71@gmail.com), 9 Principal Scientist (aktiwari63@yahoo.com). 2 Reader, Department of Biochemistry, Bundelkhand University, Jhansi (shahina.kalin@yahoo.co.in). 6 Scientist (rajivbiotech028@gmail.com), CSWRI, Avikanagar. 8 Faculty of Animal Science (aktiwari71d@gmail.com), M.J.P. Rohilkhand University, Bareilly. Cytokines are essential effector molecules of innate and acquired immunity that initiate and coordinate cellular and humoral responses aimed for eradicating pathogens. The cytokines have been successfully used in humans for the treatment of immune-deficiencies. There are various methods of cytokine administration that are currently being developed by several groups which include delivery via live vectors (viral and bacterial), DNA injection and injection of protein. The genes for chicken myelomonocytic growth factor (cMGF) (Hilton et al. 2002), stem cell factor (SCF) (Zhou et al. 1993), interleukin-8 (IL-8) (Leutz et al. 1984), type 1 interferon (Ch.IFN-) (Sekellick et al. 1994) and type 2 interferon (Ch.IFN-) (Digby and Lowenthal 1995) were cloned and recombinant protein was expressed and characterized. IFN- is a member of the interferon family of cytokines that share the property to modulate the immune response and inhibit viral replication and has anti-tumor properties. IFN- stimulates immunoglobulin production (Samuel 2001) and specific cytotoxicity of T cells (MacDonald et al. 2002). IFN-, secreted by Th-1 cells, Tc cells, dendritic cells and NK cells, is also known as immune interferon. Ch.IFN- is one cytokine that was assessed for its ability to enhance antibody response. When co-administered with antigen, recombinant Ch.IFN- produces a prolonged secondary antibody response that persists at higher levels and for longer periods compared to antigen injected alone (Lowenthal et al. 1998). Hence, it is candidate ‘Genetic adjuvants’ for humoral responses to soluble protein antigens (Schijns et al. 2000). The ability of Ch-IFN- to combat and enhance vaccine efficacy makes it an excellent candidate as a therapeutic agent and adjuvant. Therefore, in the present study, chicken IFN- was amplified and cloned in mammalian expression vector for use as ‘Genetic Adjuvant’ along with DNA vaccine. MATERIALS AND METHODS Pooled heparinized blood (2 ml) collected from 3 birds was first depleted of thrombocytes by low-speed centrifugation. The buffy coat was then layered on histopaque and centrifuged for 15 min at 800 ×g. Cells at interface were collected and washed 3 times with PBS. Viability of these cells was assessed by dye exclusion test using trypan blue stain. The cells were counted in a countess, the concentration of cells adjusted to 2×10 6 cells / ml in RPMI-1640 medium, plated in a 6 well plate and induced with 1 μg of LPS (lipopolysacchride)/ml of medium. The induced PBMC were Indian Journal of Animal Sciences 83 (4): 398–401, April 2013/Article Cloning, expression and phylogenetic analysis of IFN- gene in chicken UTTARA CHATURVEDI 1 , SHAHINA KALIM 2 , LAKSHYA VEER SINGH 3 , A P SAHOO 4 , S K PALIA 5 , RAJIV KUMAR 6 , G RAVI KUMAR 7 , SANGEETA TIWARI 8 and ASHOK K TIWARI 9 Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India Received: 23 June 2012; Accepted: 10 November 2012 ABSTRACT In the present study, chicken interferon gamma (Ch.IFN-) gene was amplified using specific primers by RT-PCR and cloned into pTZ 57R/T cloning vector. The clone was confirmed by restriction digestion analysis using KpnI and ApaI restriction enzymes and orientation was confirmed by colony PCR using T7 promoter primer and gene specific forward primers. Further, the gene fragment was sub-cloned in eukaryotic expression vector pcDNA 3.1(+) and confirmed by restriction digestion. The recombinant pcDNA 3.1 having IFN gene (pcDNA.Ch-IFN-) was checked for its ability to express IFN- in vitro in cell culture and expression was confirmed by RT-PCR, indicating the gene is functionally active. The study to evaluate ability of pcDNA.Ch-IFN- to combat infection and enhance efficacy of DNA vaccine is under way. Key words: Genetic adjuvant, IFN , RT-PCR