使用辅助生殖技术如体外受精(IVF)通过种系传播进行转基因动物的繁殖是产生用于生物医学研究的转基因菌落的最有效方法。这项研究的目的是从表达突变的人淀粉样蛋白前体蛋白(mhAPP)基因的建立犬产生转基因幼犬。实验一评估了新鲜稀释的精液的特性,游泳,和使用计算机辅助精液分析仪(CASA)的Percoll梯度方法。在Percoll梯度样品中,游动和逐渐游动的精子计数高于在游动和新鲜稀释的样品中(p<0.05)。在实验二,共有59个、70个和65个推定受精卵由新鲜,Percoll渐变,和游泳方法,分别,被转移到代孕(每组5个);Percoll梯度(27.27%)和游泳样本(14.29%)显示出最高的胚泡形成率,而新鲜稀释的精液不产生任何囊胚。实验三检查了胚胎的足月发育能力。在Percoll梯度组中的5个代理中,1只(20.0%)怀孕;它有4只(6.15%)囊,并分娩了4只(6.15%;2只雄性和2只雌性)活犬。在4只小狗中,发现2(50.0%)在GFP荧光下在其指甲和脚趾上传递转基因。此外,在所有IVF来源的幼犬的脐带中检查mhAPP转基因的整合和表达,转基因的存在仅在GFP阳性幼犬中观察到。因此,通过Percoll方法制备的精液可以使用IVF技术通过雄性种系传播产生转基因幼犬。我们的结果将有助于有效繁殖转基因狗,这将促进人类生物医学研究。
Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and
Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the
Percoll gradient samples (p < 0.05) than in the swim-up and freshly diluted samples. In Experiment II, a total of 59, 70, and 65 presumptive zygotes produced by fresh,
Percoll gradient, and swim-up methods, respectively, were transferred to surrogates (5 for each group); the
Percoll gradient (27.27%) and swim-up samples (14.29%) showed the highest blastocyst formation rates, while fresh diluted semen did not produce any blastocyst. Experiment III examined the full-term developmental ability of embryos. Among the 5 surrogates in the
Percoll gradient group, one (20.0%) became pregnant; it had 4 (6.15%) sacs and delivered 4 (6.15%; 2 males and 2 females) live puppies. Among the 4 puppies, 2 (50.0%) were found to transmit the transgene on their nail and toe under GFP fluorescence. Furthermore, the integration and expression of the mhAPP transgene were examined in the umbilical cords of all the IVF-derived puppies, and the presence of the transgene was only observed in the GFP-positive puppies. Thus, semen prepared by the Percoll method could generate transgenic puppies by male germline transmission using the IVF technique. Our result will help propagate transgenic dogs efficiently, which will foster human biomedical research.