Production of insulin in egg white by transgenic chicken

在前期的研究中，我们利用泛嗜性逆转录病毒载体构建了转基因鹌鹑，并将转基因传递给后代。在该载体中，引入了由病毒LTR启动子控制的GFP（绿色荧光蛋白）基因和由RSV启动子控制的新霉素抗性基因neo^r。利用该系统，将转基因鸡与未转基因鸡杂交，获得的G_1代鸡中80%的转基因鸡。为了实现这一点，我们将使用卵清蛋白表达和分泌系统，因为卵清蛋白是蛋清的主要蛋白质组分。首先，我们利用RCR技术从鸡基因组中克隆了卵蛋白基因，获得了3.5kb的DNA片段。将该片段与人生长因子cDNA连接后，将该表达盒插入逆转录病毒载体中，产生复制缺陷型病毒颗粒。病毒液浓缩后，将病毒颗粒注射到鸡和鹌鹑的囊胚内。卵用我们的体外培养方法孵化。经PCR检测，所有G_0代鸡均已获得转基因。G_0代雌虫仅在卵白中产生人生长激素，但量很低。用G_0代转基因鸡和非转基因鸡生产的G_1代转基因鸡，在蛋清中产生1 - 10 ng/ml的人生长激素。通过对不同器官细胞的RT-PCR分析，证明了转基因的表达具有输卵管特异性。

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.