Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.
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.
Linney
E
E
Department of Microbiology, Duke University Medical Center, Duke University, Durham, North Carolina 27710, USA.
Hardison
N L
NL
Lonze
B E
BE
Lyons
S
S
DiNapoli
L
L
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D017931:DNA Primers; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins