Plant & Animal Genomes XIV Conference Plant & Animal Genomes XIV ConferenceJanuary 14-18, 2006
Town & Country Convention Center
San Diego, CA
J. N. Petitte , Q. Wu , S.L. Pardue , S. Borwornpinyo , C. Giamario , P.E. Mozdziak
Transgenic chickens were produced using an SNV-based vector carrying a nuclear-localized lacZ, and birds expressing bacterial beta-galactosidase were generated (Mozdziak et al. 2003, Dev. Dyn. 226: 439-445). Further characterization of beta-galactosidase expression and complete detailing of the proviral insertion event has now been performed on the transgenic birds. Genomic sequences flanking the retroviral insertion site were identified using inverse-PCR, homozygous individuals were identified using PCR-based genotyping, and beta-galactosidase expression was evaluated using Western analysis and histochemistry. Based upon the current draft of the chicken genome, the viral insertion carrying the lacZ gene was found on chromosome 11 within intron 1 of the predicted gene for fractalkine/neurotactin (CX3CL1). RT-PCR analysis of fractalkine expression in wild-type birds confirmed the predicted genomic structure. When Generation 2 (G2) lacZ -positive hemizgous birds were inter-mated to generate 361 G3 progeny, 97 (26.9%) were wild-type non-transgenic, 182 (50.4%) were hemizygous for lacZ , and 82 (22.7%) were homozyous for lacZ. Histochemical analysis revealed beta-galactosidase activity in most tissues, and western analysis suggests the highest expression in the muscle and liver. In addition, it appears that fractalkine mRNA expression is absent from the brain of homozygous transgenic individuals, suggesting that the birds are also an insertional knockout for fractalkine. With the identification of homozygous birds, the line is now designated NCSU-Blue1.