Chrenek P, Bauer M, Makarevich A V
Animal Production Research Centre Nitra, Hlohovecka 2, 951 41 Luzianky, Nitra, Slovak Republic.
Zygote. 2011 Feb;19(1):85-90. doi: 10.1017/S0967199410000109. Epub 2010 May 28.
The aim of this study was to compare the quality of rabbit transgenic embryos obtained upon microinjection of gene constructs containing different promoters and green fluorescent proteins (CMVIE-EGFP, PGK-EGFP and CMVIE-hrGFP). Developmental rate, total cell number in hatching blastocyst stage, number of apoptotic cells, diameter of embryos, transgene integration and transgenic mosaicism were investigated.The rate of rabbit embryos microinjected with the different gene constructs developed up to morula stage was significantly lower (p < 0.05) than that of intact (non-microinjected) rabbit embryos (66-74vs. 98%). The highest efficiency of transgene integration (15%) was found when the CMVIE-EGFP (DrdI) gene construct was used, however a significantly higher transgenic mosaicism (60%) was found in rabbit embryos using this gene. The lowest cell number was counted in rabbit transgenic embryos with CMVIE-rhGFP linearized by ScaI (115.0 ± 8.20), the highest cell number (134.0 ± 35.00) was detected in rabbit transgenic embryos carrying PGK-EGFP (Not I) gene. The highest number of apoptotic cells (2.6 ± 0.33) was recorded in rabbit transgenic embryos with the integrated CMVIE-EGFP (ClaI) transgene.Based on these results a more suitable gene marker for rabbit transgenic embryos production and selection is the CMVIE-EGFP (ClaI) gene construct. Prior to using microinjected embryos (for embryo transfer, vitrification or ESC isolation) it is necessary to pre-select microinjected embryos with evident transgenic mosaicism.
本研究的目的是比较通过显微注射含有不同启动子和绿色荧光蛋白（CMVIE-EGFP、PGK-EGFP和CMVIE-hrGFP）的基因构建体所获得的兔转基因胚胎的质量。研究了发育率、孵化囊胚阶段的总细胞数、凋亡细胞数、胚胎直径、转基因整合和转基因嵌合现象。显微注射不同基因构建体的兔胚胎发育至桑椹胚阶段的比率显著低于完整（未显微注射）兔胚胎（66 - 74%对98%，p < 0.05）。当使用CMVIE-EGFP（DrdI）基因构建体时，发现转基因整合效率最高（15%），然而使用该基因的兔胚胎中发现转基因嵌合现象显著更高（60%）。用ScaI线性化的CMVIE-rhGFP兔转基因胚胎中细胞数最少（115.0 ± 8.20），携带PGK-EGFP（Not I）基因的兔转基因胚胎中检测到的细胞数最多（134.0 ± 35.00）。整合有CMVIE-EGFP（ClaI）转基因的兔转基因胚胎中记录到的凋亡细胞数最多（2.6 ± 0.33）。基于这些结果，用于兔转基因胚胎生产和选择的更合适的基因标记是CMVIE-EGFP（ClaI）基因构建体。在使用显微注射胚胎（用于胚胎移植、玻璃化或胚胎干细胞分离）之前，有必要预先选择具有明显转基因嵌合现象的显微注射胚胎。
