Feng Hao, Zeng Zhi-Qiang, Liu Shao-Jun, Zhang Xuan-Jie, Zhou Gong-Jian, Li Jian-Zhong, Liu Yun, Wang Ya-Ping, Chen Shang-Ping, Hu Wei, Zhu Zuo-Yan
College of Life Science, Hunan Normal University, Changsha 410081, China.
Yi Chuan Xue Bao. 2002 May;29(5):434-7.
The tetraploid fish has been developed by assortative breeding the hybrids of Carassius auratus red var. (Female) x cyprinus carpio (Male), which has the stable genetic characters and can reproduce themselves. An "all-fish" recombinant DNA construct (pCAgcGHc) containing common carp beta-actin gene promoter and cDNA for grass carp (Ctenopharyngodon idella) growth hormone gene was introduced into fertilized eggs of the allotetroploid fish through microinjection as soon as artificial insemination was done. Artificial insemination was carried out between the female and the male transgenic allotetraploid fish which contain the "all-fish" recombinant DNA construct (pCAgcGHc) and are the biggest in the size. Fifty F1 samples of transgenic allotetraploid fish of 150 days and 50 allotetraploid fish (regarded as the control) were chosen, and the weight and the body length of each were measured, the results showed that F1 of transgenic allotetraploid fish of 150 days had obvious growth dominance compared with the control. Genomic DNA of tail fin was extracted from 20 F1 of transgenic allotetraploid fish of 150 days and the control. Proper primers were introduced to check whether the sample had the transgene. Pa, the upstream primer, is located in beta-actin promoter, and Pg, the downstream primer, is located in growth hormone cDNA for grass carp (gcGHc). The transgene was detected in 90% F1 of transgenic allotetraploid fish in tail fin DNA by polymerase chain reaction (PCR) amplification. Sperm could be squeezed out from a few F1 of transgenic allotetraploid fish of 150 days, however, this phenomenon did not exist in the controls. The importance of forming the pure line of transgenic allotetraploid was elucidate in the paper.
四倍体鱼是通过对红鲫（雌）×鲤鱼（雄）的杂种进行选择性育种培育而成的，具有稳定的遗传特性且能自我繁殖。在人工授精完成后，立即通过显微注射将含有鲤鱼β - 肌动蛋白基因启动子和草鱼（Ctenopharyngodon idella）生长激素基因cDNA的“全鱼”重组DNA构建体（pCAgcGHc）导入异源四倍体鱼的受精卵中。在含有“全鱼”重组DNA构建体（pCAgcGHc）且体型最大的转基因异源四倍体雌鱼和雄鱼之间进行人工授精。选取50尾150日龄的转基因异源四倍体鱼样本和50尾异源四倍体鱼（作为对照），测量每尾鱼的体重和体长，结果表明，150日龄的转基因异源四倍体鱼F1与对照相比具有明显的生长优势。从20尾150日龄的转基因异源四倍体鱼F1和对照的尾鳍中提取基因组DNA。引入合适的引物来检测样本是否含有转基因。上游引物Pa位于β - 肌动蛋白启动子中，下游引物Pg位于草鱼生长激素cDNA（gcGHc）中。通过聚合酶链反应（PCR）扩增，在90%的150日龄转基因异源四倍体鱼F1的尾鳍DNA中检测到了转基因。从少数150日龄的转基因异源四倍体鱼F1中可以挤出精子，然而，对照中不存在这种现象。本文阐述了形成转基因异源四倍体纯系的重要性。
