To develop a transgenic line for conditional expression of desired genes in rats, we used the tetracycline regulatory system and generated several lines of the transgenic rats carrying the tTA transgene. Using a vigorous and ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in the central nerve system, skeletal muscle, heart, lung, and in most abdominal organs. Expression of the tTA was sufficient to vigorously activate its reporter gene, but was below the threshold to produce toxicity to the rats. These tTA transgenic rats are a model of inducible and reversible gene expression in the rat, and could be valuable to the development of genetic rat models for human diseases.
The Tet-regulatory system is widely used to achieve inducible and reversible expression of desired genes in vivo
. To date, a great number of transgenic mice have been generated to express tTA (Tet-off) or rtTA (Tet-on) transactivators in ubiquitous or tissue-specific patterns 26, 27, 39
. While few lines of transgenic rats have been created to express tTA in cell-specific patterns 29, 40, 41
, a single line of tTA or rtTA transgenic rat is not available for ubiquitous activation of reporter gene in various cell types. To obtain ubiquitous expression of the tTA transgene in rats, we chose the ubiquitous promoter CAG, a hybrid promoter that has been constructed by fusing the cytomegalovirus enhancer with the chicken beta actin promoter. CAG has been shown to produce ubiquitous and robust gene expression in transgenic mice 35, 42, 43
. Although CAG is a vigorous promoter, expression of tTA protein was not detectable by immunoblotting in the two transmittable lines (data not shown). We used the original bacterial codon for tTA. Sanbe et al. reported that bacterial codon usage bias leads to alternative splicing of tTA mRNA and results in a low abundance of full-length tTA mRNA 39
. As a transcriptional activator, tTA at limited levels should be sufficient to activate its target gene. Excessive expression of tTA protein may cause toxicity to the cells because high abundance of exogenous transactivator may interfere with cellular transcription activity.
Indeed, enhanced expression of tTA using mammalian codon causes toxicity in transgenic mice 39, 44
. To balance ubiquitous with adequate gene expression, we chose a ubiquitous promoter as the driver, but used the original bacterial codon for tTA. Consistent with previous finding in transgenic mice 26, 35, 39, 42, 43
, our results in transgenic rats demonstrated that expression of tTA with bacterial codon was under the detection threshold by western blotting, but was sufficient to vigorously activate its target gene in various tissues. Insertion or expression of the tTA transgene may interfere with endogenous genes and cause toxicity to the animals. If the toxicity of the tTA transgene is evident, the toxicity should be reflected as an effect on growth, overall health, or organ development. In the two transmittable lines of transgenic tTA rats, no detectable abnormality was observed. These results suggest that insertion of the tTA transgene did not result in a deleterious dominant mutation and that expression of the tTA transgene did not obviously interfere with endogenous genes. These transgenic rats express tTA within a safe dosage and at sufficient levels to activate the target gene.
The dynamics of Dox-regulated gene expression were thoroughly examined for the first time in transgenic rats. We not only examined suppression of tTA-activated gene expression by Dox, but also examined reversibility of tTA-dependent gene expression after Dox withdrawal. Both the suppression and the reversal of gene expression were tested in adult and neonatal rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subjected to the regulation by Dox. In transgenic mice, Dox dose-dependently suppresses tTA-activated gene expression and washout of Dox remnant effect also corresponds to Dox dosage used 26, 39
. Similarly, Dox reached its maximal effect of gene suppression at speeds proportional to its dose. High dose of Dox (500 μg/ml in drinking water) took a short time to fully suppress tTA-activated gene expression, but required a much long time to remove its remnant effect. In our studies, a duration of 5 days appeared to be the minimal time for complete gene suppression by orally administered Dox. We tested a complex regime of Dox administration to quickly reach the maximal effect but to successfully avoid extended duration of Dox cleanout. Dox was used at a high dose (500 μg/ml in drinking water) for 2 days to reach its effective concentration quickly and then used at a low dose (20 μg/ml) to maintain its effect constantly. This improved regime of Dox administration can help achieve a quick switch of tTA-activated gene expression between ON and OFF status. In addition, parental administration of Dox fully suppressed tTA-activated gene expression in offspring rats. Milk-secreted Dox was sufficient to reach its effective concentration in neonatal rats. Parental administration of Dox can help provide a constant suppression of tTA-dependent gene expression during embryonic and postnatal development. We also tested the tTA transgenic lines with another reporter gene and observed similar results (data not shown).
Although laboratory mice are preferred for functional genetics, laboratory rats show significant advantages as model animals in pharmacological and toxicological studies, in behavioral analysis, in microsurgery such as cell transplantation, and in studies on neurological diseases. In modeling some human diseases, animal species may play an important role in phenotypic expression. For example, no substantial loss of dopaminergic neurons is observed in human alpha-synuclein transgenic mice 45, 46
, but progressive loss of the neurons is induced by transient expression of human alpha-synuclein in rats 47
. Animal species may not be a sole factor contributing to the variation of phenotypic expression in alpha-synuclein models. In some genetic models for human disease, developmental compensation may dilute phenotypic expression when the gene is constitutively mutated. Such compensatory changes are increasingly observed in genetic models 48, 49
. Disrupting one gene of the JNK family causes compensatory changes in the other related genes 49
. Studies on Jnk2
knockout mice with the gene disrupted from germline suggest a negative regulatory role in cell proliferation for the protein kinase Jnk2 50
. In contrast, studies of Jnk2
inhibition in adult mice suggest Jnk2 as a positive regulator for cell proliferation 49
. These distinct phenotypes may result from developmental compensation by upregulated expression of the related genes 49
. Compared with the human, the rodent has a short lifetime. Developmental compensation may buffer the detrimental effects of gene mutation and thus attenuate or even prevent the phenotypes of gene mutation from developing in a rodent's lifetime. Selective expression of disease genes in adult animal may increase the chance to reproduce the phenotypes of human diseases in the animals. Recent advances in transgenic RNAi indicates that RNAi-mediated gene silencing can reproduce phenotypes of the gene knockout 37, 51, 52
. Transgenic RNAi is a convenient approach to achieve hypomorphic phenotypes in the transgenic rats. To maintain RNAi transgenic lines, conditional expression of the RNAi transgene is necessary when the hypomorphic phenotype is lethal or infertile. In the aforementioned applications, the tTA transgenic rats would be a valuable tool for accomplishment of these goals.