Hsiao C D, Hsieh F J, Tsai H J
Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.
Dev Dyn. 2001 Apr;220(4):323-36. doi: 10.1002/dvdy.1113.
Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.
转基因在F0代中的嵌合表达严重阻碍了对转基因鱼瞬时表达的研究。为避免嵌合现象，构建了增强型绿色荧光蛋白（EGFP）基因盒并将其导入单细胞斑马鱼胚胎中。这些EGFP基因盒两侧是来自腺相关病毒（AAV）的反向末端重复序列（ITR），并由斑马鱼α-肌动蛋白（pα-肌动蛋白-EGFP-ITR）或青鳉β-肌动蛋白启动子（pβ-肌动蛋白-EGFP-ITR）驱动。在注射pα-肌动蛋白-EGFP-ITR的存活者中，56%±8%的个体其骨骼肌中EGFP呈特异性且均匀表达；在注射pβ-肌动蛋白-EGFP-ITR的存活者中，1.3%±0.8%的个体其全身均有EGFP表达。在注射未侧翼连接AAV-ITR的EGFP基因的斑马鱼胚胎中，从未出现过均匀的瞬时表达。在F0代中，均匀分布的EGFP在发育早期可模拟转基因品系中的稳定表达。我们建立了5个源自与野生型鱼杂交的注射pα-肌动蛋白-EGFP-ITR胚胎的转基因品系，以及11个源自与野生型鱼杂交的注射pβ-肌动蛋白-EGFP-ITR胚胎的转基因品系。通过聚合酶链反应分析证实，这些转基因品系均未出现转基因不表达的情况。α-肌动蛋白和β-肌动蛋白转基因品系均实现了转基因的稳定孟德尔遗传，且不改变表达和整合模式。子代遗传试验和Southern印迹分析结果强烈表明，侧翼连接AAV-ITR的转基因以串联倍增体形式随机整合到基因组的单个位点。因此，将AAV-ITR整合到转基因中可导致F0代中基因的均匀表达以及斑马鱼中转基因的稳定传递。
