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.

Cloning of rainbow trout (Oncorhynchus mykiss) α-actin,myosin regulatory light chain genes and the 5′-flanking region ofα-tropomyosin. Functional assessment of promoters

“…4E) were located in this structure. Interestingly, YSL is characterized by an exclusively high capacity for transgene expression (Williams et al, 1996). In all analysed embryos, LacZ-positive cells were detected in neural tissues.…”

Regulation and expression of transgenes in fish—a review

“…Although widely attributed to unequal distribution of transgene copies (see reviews by Houdebine and Chourrout, 1991;Fletcher and Davies, 1991), it appears that such mosaicism with respect to expression may also partly be a result of differential expressivity of the transgene within cells of the same embryo, as observed by Kothary et al (1992) in mouse embryos. Furthermore, differential replication of transgene copies in multinucleated or polyploid tissues such as muscle cells or the yolk syncytial layer (YSL) cells may explain additional variability in transgene expression patterns with higher levels consistently detected in these tissues (Williams et al, 1996).…”

Structure and Functional Analysis of a Tilapia (Oreochromis mossambicus) Growth Hormone Gene: Activation and Repression by Pituitary Transcription Factor Pit-1

“…Note that in some embryos, there was an intense expression in the yolk sac endoderm . This ectopic high-level transgene expression was probably attributable to the differential replication of transgene copies in multinucleated or polyploid tissues such as the yolk syncytial layer (Williams et al, 1996). In the rat, Pit-1 autoregulates its own expression by binding to two sites.…”