Linney E, Hardison N L, Lonze B E, Lyons S, DiNapoli L
Department of Microbiology, Duke University Medical Center, Duke University, Durham, North Carolina 27710, USA.
Dev Biol. 1999 Sep 1;213(1):207-16. doi: 10.1006/dbio.1999.9376.
To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.
为评估将表达转基因导入斑马鱼生殖系的替代方法，使用假型逆转录病毒载体感染和胚胎DNA显微注射技术，培育出了表达核靶向增强型绿色荧光蛋白（eGFP）基因的转基因鱼。鉴定出了生殖系转基因奠基者，并对这些奠基者的胚胎后代进行了eGFP表达程度和模式的评估。为比较两种转基因模式，两种载体均使用了非洲爪蟾翻译延伸因子1-α增强子/启动子调控元件。鉴定出了数条将eGFP表达传递给后代的转基因奠基鱼。描述并比较了两种基因转移模式下的基因表达模式。通过DNA显微注射或逆转录病毒载体感染导入转基因后1天，检测到了eGFP的瞬时表达。在两种基因转移情况下，转基因雌性在合子基因组激活之前就产生了eGFP阳性后代。因此，在受精前卵母细胞就提供了GFP。一只转基因雌性在其卵巢卵泡中显示出eGFP表达。合子基因表达诱导后，转基因后代胚胎中基因表达的定性模式相似，且与转基因模式无关。受精后5-7小时内可检测到新合成的GFP的出现。DNA注射转基因的转基因奠基者之间eGFP表达程度的变异性比逆转录病毒载体产生的转基因更大。通过逆转录病毒载体感染提供表达生殖系转基因后代的能力，既为斑马鱼研究提供了一种替代转基因模式，也为轻松评估逆转录病毒载体感染斑马鱼胚胎的插入诱变频率提供了一种可能的方法。然而，由于GFP从卵母细胞转移到胚胎，GFP的稳定性可能会给像斑马鱼胚胎那样快速发育的胚胎分析带来问题。
