Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

Abstract

The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human β‐actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16‐responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre‐implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2‐cell to the 4‐cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility. Mol. Reprod. Dev. 55:37–46, 2000. © 2000 Wiley‐Liss, Inc.

A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet‐on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism

“…Basal uninduced tyrosinase expression was also observed (Figs 2 and 3) in TRETyBS transgenic mice (mainly confined at central regions of the retina) indicating background residual activity, as reported in similar experiments (16)(17)(18)(19)(20)37). To compensate for the deficits associated with standard TET-ON inducible schemes, observed here, new chimeric transactivation proteins have been produced either containing just an optimization of the codon usage (ÔmammalianizedÕ, 16,20), or a combination of codon optimization and new mutations proposed to reduce the basal activity of rtTA while increasing its inducibility (41), whose activity is being explored in vitro and in vivo (41,42).…”

“…The level of transactivation achieved with the four germ‐line transmitting HSTyrTET transgenic mouse lines, firstly assessed in combination with the L7 reporter transgenic mice and secondly established with transgenic mice carrying the TRETyBs construct, was relatively weak. A number of explanations can be provided for this, including: 1) the likely selection occurring in utero for transgenic founders either displaying low levels of expression of the toxic rtTA protein (17), or being mosaic (most, ∼69%, of HSTyrTET lines did not transmit the transgene through the germ‐line), 2) the reported difficulty in generating transgenic mouse lines expressing high levels of rtTA (TET‐ON), recognized as one of the major limitations of this system, as compared with equivalent mouse lines made with tTA (TET‐OFF) (37), 3) the fact that the coding region for the rtTA protein derives, in part, from a prokaryotic genome, shown to impair adequate transcription in mammalian genomes (38). In this regard, the artificial combination of tyrosinase regulatory elements, including the LCR and the promoter of the mouse tyrosinase gene, shown to work in other experimental designs at different levels, not necessarily reproducing the endogenous pattern for the tyrosinase gene (39), could have compromised the actual expression levels of the HSTyrTET construct, therefore only those lines with limited and more restricted (only RPE cells, but not neural crest‐derived melanocytes) rtTA gene expression have eventually been detected and established.…”

Conditional control of gene expression in the mouse

“…Here, I review these systems and discuss certain studies that exemplify the advantages and limitations of each system. due to its interaction with mouse transcription factors 8,9 . Squelching might also occur when chimeric transactivators that contain the VP16-activating domain are highly expressed, and in vitro solutions to this problem have been reported 9,10 .…”

Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice

“…A single Gal4-DBD transgenic founder line was also generated as a control. Previous studies showed that Gal4-VP16 expression was detrimental to early mouse development (6), and SV40 T-antigen expression driven by the lck proximal promoter induced thy-http://bmbreports.org BMB reports Fig. 2.…”

“…However, when expressed at high levels the Gal4-VP16 transactivator was shown to inhibit transcription by binding and titrating out transcription factors (5). Furthermore, the VP16 protein has been shown to be toxic to pre-implantation mouse embryos (6). Therefore, achieving a balance between transactivation and toxicity will be required to attain optimal results from this system.…”