Production of insulin in egg white by transgenic chicken

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In previous study, we used pantropic retrovirus vector and constructed transgenic quails that transmitted transgene to offsprings. In this vector, GFP (Green fluorescence protein) gene controlled by virus LTR promoter and Neomycin resistant gene neo^r controlled by RSV promoter were incorporated. By this system, 80% of G_1 offsprings which were produced by crossing transgenic G_0 and no transgenic birds, contained the transgene.For the practical application of transgenic bird, accumulation of useful proteins in eggwhite is essential. To attain this, we will use ovalbumin expression and secretion system, since ovalbumin is a major protein component of eggwhite. First, we molecularly cloned ovalbumn gene by RCR from chicken genome and obtained 3.5kb DNA fragment. After ligating this fragment with human-growth factor cDNA, this expression cassette was inserted in the retrovirus vector, and replication defective virus particles were produced. After condensation of virus solution, virus particles were injected in blastderm of chicken and quail. Eggs were hatched by our in vitro culture method. The presence of transgene was confirmed by PCR in all G_0 birds. Furthermore, human growth hormone was produced by the G_0 females exclusively in eggwhite but the amount was very low. G_1-transgenic bird produced by G_0-transgenic and non-transgenic birds produced l-10ng/ml human-growth hormone in eggwhite. By the RT-PCR analyses of the cell samples from various organs, it was shown that the expression of the transgene was oviduct-specific.