Production of insulin in egg white by transgenic chicken

在以前的研究中，我们利用泛嗜性逆转录病毒载体，构建了将转基因基因传递给后代的转基因鹌鹑。将病毒启动子控制的绿色荧光蛋白(GFP)基因和RSV启动子控制的新霉素抗性基因neo？r整合到该载体中。在该系统中，转基因鸡与非转基因鸡杂交产生的G1代后代中有80%含有转基因基因，为转基因鸡的实际应用，蛋清中有用蛋白的积累至关重要。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成分。首先，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基因连接后，插入逆转录病毒载体，得到复制缺陷病毒颗粒。病毒溶液浓缩后，将病毒颗粒注射到鸡和鹌鹑的胚泡中。用我们的体外培养方法孵化出卵子。经聚合酶链式反应证实，所有G0代家禽中均存在转基因。此外，人类生长激素只在蛋清中由G_0雌性产生，但量很少。转G_0基因鸡和非转基因鸡产生的G_1代鸡在蛋清中产生10 ng/ml的L人生长激素。通过对不同器官细胞样品的RT-PCR分析，表明转基因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.