Zhang Q, Lin J, Yu Q H, Hu W W, Yang Q
College of Veterinary, Nanjing Agricultural University, Weigang, Jiangsu, China.
College of Veterinary, Nanjing Agricultural University, Weigang, Jiangsu, China
Genet Mol Res. 2015 Mar 20;14(1):2006-14. doi: 10.4238/2015.March.20.10.
Transgenic goats have been utilized for years to produce valuable protein. However, when transgenic goats are produced by random integration of inserted genes into cells, the copy number and integration sites of these genes in the goat genome are typically indefinite. Most polymerase chain reaction (PCR)-based methods that have been utilized to determine copy number and integration sites of inserted genes in the genome require complicated manipulations. In this study, we used quantitative real-time PCR and thermal asymmetric interlaced-PCR to determine copy number and integration sites of the inserted genes, respectively. Copies of transgenic goat lines GHcd-2 and GHcd-7 were 12.95 ± 0.18 and 12.24 ± 1.12, respectively. Two integration sites, located in chromosomes 3 and 11 and referred to as tg1 and tg2, were identified by thermal asymmetric interlaced-PCR. Junction PCR was then performed to confirm the integration sites of growth hormone transgenic goats. Transgenic copy number and integration sites were determined, which will be useful for determining the relationship between the growth hormone expression, copy number, and integration sites.
转基因山羊已被用于生产有价值的蛋白质多年。然而，当通过将插入基因随机整合到细胞中来生产转基因山羊时，这些基因在山羊基因组中的拷贝数和整合位点通常是不确定的。大多数用于确定基因组中插入基因拷贝数和整合位点的基于聚合酶链反应（PCR）的方法都需要复杂的操作。在本研究中，我们分别使用定量实时PCR和热不对称交错PCR来确定插入基因的拷贝数和整合位点。转基因山羊品系GHcd - 2和GHcd - 7的拷贝数分别为12.95±0.18和12.24±1.12。通过热不对称交错PCR鉴定出两个位于3号和11号染色体上的整合位点，分别称为tg1和tg2。然后进行连接PCR以确认生长激素转基因山羊的整合位点。确定了转基因拷贝数和整合位点，这将有助于确定生长激素表达、拷贝数和整合位点之间的关系。
