title: High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.
abstract: For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.
authors.affiliation: Department of Biology, School of Biological Sciences, University of Southampton, UK.
authors.forename: D W
authors.initials: DW
authors.lastname: Williams
authors.forename: F
authors.initials: F
authors.lastname: Müller
authors.forename: F L
authors.initials: FL
authors.lastname: Lavender
authors.forename: L
authors.initials: L
authors.lastname: Orbán
authors.forename: N
authors.initials: N
authors.lastname: Maclean
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D002467:Cell Nucleus / Q000235:genetics; D003845:Deoxycytosine Nucleotides / Q000235:genetics; D004530:Egg Yolk / Q000378:metabolism / Q000648:ultrastructure*; D004625:Embryo, Nonmammalian / Q000648:ultrastructure; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000737:chemistry; D015726:Giant Cells; D008297:Male; D008845:Microinjections; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D003845:Deoxycytosine Nucleotides; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; C024107:2'-deoxycytidine 5'-triphosphate; D001616:beta-Galactosidase