Chan A W, Kukolj G, Skalka A M, Bremel R D
Endocrinology-Reproductive Physiology Program, University of Wisconsin-Madison, USA.
Mol Reprod Dev. 1999 Apr;52(4):406-13. doi: 10.1002/(SICI)1098-2795(199904)52:4<406::AID-MRD9>3.0.CO;2-P.
Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.
在胚胎移植前对转基因胚胎进行筛选是提高转基因家畜生产效率的一种手段。在转基因报告基因中，绿色荧光蛋白（GFP）的细胞质表达具有使其成为转基因胚胎筛选理想选择的特征。本研究的主要目的是评估一种特殊设计的GFP报告基因的细胞质表达作为转基因牛胚胎筛选工具的效果。第二个目的是评估该报告基因用于研究与原核显微注射DNA整合时间相关的转基因嵌合体。通过原核注射产生的转基因胚胎显示出GFP表达的离散模式，分别有25%、50%和100%的卵裂球表达GFP形成簇状。这种嵌合模式被解释为表明显微注射DNA的整合不仅发生在原核阶段，也发生在随后的细胞分裂中。在GFP阳性的转基因胚胎中，只有21%的胚胎所有卵裂球都显示绿色荧光。利用胚胎内阳性卵裂球的比例，估计了显微注射DNA的整合时间。表达GFP的非嵌合胚胎的频率与已发表的来自原核显微注射胚胎的转基因牛种系传递成功率一致。这些结果表明GFP作为转基因胚胎标记物的可能应用，并直观地说明了原核显微注射胚胎中DNA整合的潜在复杂性。
