Burdon T G, Wall R J
Beltsville Agricultural Research Center, U.S. Department of Agriculture, Maryland 20705.
Mol Reprod Dev. 1992 Dec;33(4):436-42. doi: 10.1002/mrd.1080330410.
The state of genes microinjected into mouse embryos was followed from the one-cell to the blastocyst stage using the polymerase chain reaction (PCR). Microinjected DNA was detected in all one-, two-, and four-cell injected embryos and in 44% of morula and 26% of blastocysts. Head-to-tail ligation of microinjected genes, a common feature of stably integrated transgene arrays, was detected in all embryos after injection of microinjected genes and occurred irrespective of the structure at the ends of the injected genes. Sensitivity of microinjected DNA to a methylation-dependent restriction endonuclease Dpn I was lost in all embryos by the two-cell stage (24 hr), indicating a change in DNA methylation, independent of transgene integration. Dissociation of blastomeres prior to compaction revealed a mosaic distribution of the microinjected DNA within the embryo and supports the notion that injected genes form a limited number of arrays, which segregate independently until they integrate into the genome or are degraded.
利用聚合酶链反应（PCR），追踪显微注射到小鼠胚胎中的基因从单细胞期到囊胚期的状态。在所有注射过的单细胞、二细胞和四细胞胚胎中均检测到显微注射的DNA，在桑椹胚中有44%检测到，在囊胚中有26%检测到。稳定整合的转基因阵列的一个共同特征——显微注射基因的首尾连接，在显微注射基因后在所有胚胎中均被检测到，且与注射基因末端的结构无关。到二细胞期（24小时）时，所有胚胎中显微注射的DNA对甲基化依赖性限制性内切酶Dpn I的敏感性均丧失，这表明DNA甲基化发生了变化，与转基因整合无关。在致密化之前对卵裂球进行解离，揭示了显微注射的DNA在胚胎内的镶嵌分布，并支持这样一种观点，即注射的基因形成数量有限的阵列，这些阵列独立分离，直到它们整合到基因组中或被降解。
