Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

Construction of transgenic DNA and creation of transgenic rats

A Tet-off system was chosen to produce inducible and reversible gene expression in transgenic rats. This system is readily used in transgenic mice ^26-29. The Tet-off system is comprised of two elements: a tetracycline-controlled transactivator (tTA) and a tTA-dependent promoter. The tTA-dependent promoter is a hybrid promoter that is constructed by juxtaposing multiple tetracycline-responsive elements (TRE) upstream of a minimal cytomegalovirus promoter (TREminiCMV). When a gene is placed under control of the TREminiCMV promoter, transcription of the gene depends on activation by the transcriptional activator tTA. The tTA activates its reporter gene in the absence of tetracycline or tetracycline derivatives such as Dox (Dox), but is inactivated in the presence of Dox. For robust gene expression, a strong hybrid promoter, CAG (cytomegalovirus enhancer fused to chicken beta actin promoter), was used to drive a tTA gene that was extracted from the vector pTet-off (Clontech) ^{35, 36}. The transgenic construct CAG-tTA consisted of the CAG promoter, the noncoding exon along with the first intron of the beta actin gene, tTA cDNA, and three repeats of the SV40 polyadenylation signal (Figure 1A). The human Leucine Rich Repeat Kinase 2 (LRRK2) gene tagged with hemaglutinin (HA) was driven by the TRE promoter and used as the reporter gene for tTA (Figure 2A).

Both the CAGtTA and the TREhLRRK2-HA transgenic constructs were linearized by restriction digestion and purified from an agarose gel. The transgene DNA was injected into the pronucleus of fertilized single-cell embryos of Sprague-Dawley (SD) rats. Surviving eggs were transferred into pseudopregnant female rats. Transgenic founder rats were identified by PCR-magnifying part of the transgenes with the following primers: 5'-AGCCTCTGCTAACCATGTTC-3' (forward) and 5'-AACCTTCGATTCCGACCTCA-3' (reverse) for the tTA transgene, and 5'-CTGTTGATCGTCTTGGACTC-3' (forward) and 5'-CCCAATCATTTCCAACATCC-3' (reverse) for the report gene hLRRK2. Copy number of the transgenes was determined by quantitative PCR. Transgenes were maintained on the genomic background of SD rats. Each line of the tTA transgenic founders was crossed with an expressing line of the hLRRK2-HA transgenic rats to produce double transgenic offspring (F1), in which the transcriptional activity of tTA was examined. Animal use followed NIH guidelines. The animal use protocol was approved by the Institutional Animal Care and Use Committees (IACUC) at Thomas Jefferson University.

Doxycycline treatment

To suppress tTA-activated gene expression, doxycycline hydrochloride (Dox; a Sigma-Aldrich) was continually supplied in drinking water at desired concentrations. Dox-containing water was supplied in a light-protected bottle and was renewed every three days. Rats were given regular food and water supplied with or without Dox of the indicated concentrations.

Quantitative PCR

Copy number of the transgene was estimated by quantitative PCR. Genomic DNA was extracted from tail biopsy and was adjusted to a constant concentration of 100 ng/μl. A pair of primers was selected specifically for the tTA transgene: 5'-AGCCTCTGCTAACCATGTTC-3' (forward) and 5'-AACCTTCGATTCCGACCTCA-3' (reverse). In parallel, the rat tyrosine hydroxylase (TH; a known single-copy gene) gene was used as an internal control for PCR quantification and was magnified with the primers 5'-CTCAAGAATCCTGTCACCAG-3' (forward) and 5'-ACTGCCTTTCAGGGTATGTC-3' (reverse). Rat genomic DNA was estimated to contain 3 x 10⁹ base-pairs. The tTA transgene DNA was dissolved in genomic DNA of nontransgenic rats to obtain a series of standards from one to ten copies per genome. The resulting standard curve of tTA gene copy number was used for estimation of tTA transgene copy number in individual transgenic lines.

The mRNA level of the reporter gene human LRRK2 was also estimated with quantitative PCR. Total RNA was isolated from rat tissue with Trizol reagent (Sigma) and was further purified by digesting genomic DNA on a column (RNA easy kit and RNase-free DNase: Qiagen). One microgram of the purified RNA from each sample was reversely transcribed to cDNA with oligo-dT primer (RT kit, Invitrogen). Relative levels of hLRRK2 mRNA in individual samples were estimated by quantitative PCR with the following gene-specific primers: 5'-CAGCCTGACTATCAACTTCT-3' (forward) and 5'-TTGTTTGTGGATCGCTGTGA-3' (reverse) for the reporter gene hLRRK2, and 5'-TGGTTCGCTACTCCCTTGAC-3' (forward) and 5'-CTTGATGGCCTGGGCAGTT-3' (reverse) for rat L17 gene. The mRNA level of the L17 gene was used as an internal control for PCR quantification.

For quantitative PCR, the gene-specific primers were used at a concentration of 500 nM. Gene doses were measured by quantitative PCR with SYBR green kit per manufacturer's instruction (Qiagen). Cycling conditions were 15 min at 95°C followed by 40 cycles of 15 seconds at 94°C, 30 seconds at 60°C, and 20 seconds at 72°C. Aliquots of the amplified products were separated on 3% agarose gels to ensure amplification of the specific products at the predicted length. The threshold cycle number (Ct) for an examined gene was normalized to the Ct for the individual internal genes: TH gene for estimation of tTA transgene copy and L17 gene for estimation of reporter gene mRNA. The relative mRNA level of the reporter gene hLRRK2 was determined and expressed as a ratio of the mRNA level in the Dox-treated rats to that in the Dox-untreated rats, as described previously ³⁷.

Western blotting

Animal tissues were homogenized in phosphate buffer (pH 7.4) supplied with protease inhibitors (Sigma). Tissue debris, unbroken cells, and cell nuclei were removed by centrifugation at 16,000 × g for 10 minutes. Protein content in the cleared lysate was determined by BCA assay (BioRad). Since protein product of the reporter gene human LRRK2 was about 280 kDa, the LRRK2 protein was barely detected in completely denatured sample ³⁸. Protein sample of certain amounts was mixed with 2% SDS-containing loading dye and was partially denatured by incubating at 60°C for 5 minutes. Proteins were separated on 4-20% gradient SDS-PAGE and blotted onto GeneScreen Plus membrane (Perkin Elmer). Immunoreactivity to the epitope HA was detected by incubating the membrane with rabbit polyclonal antibody to HA (Rockland Inc., Pennsylvania). Immunoreactivity to GAPDH was also detected by incubating the membrane with a mouse monoclonal antibody to GAPDH (Abcam) and used as a control for equal loading. The immunoreactivity signal was developed with Super Signal kit (Pierce).

Immunohistochemistry

Rats were transcardially perfused with 4% paraformaldehyde in 0.1 M ice-cooled phosphate buffer. Their brains were dissected and further fixed in the same fixative for additional 24 hours. The dissected tissues were dehydrated in 30% sucrose and cut into serial coronal sections (30 μm thick) on a Cryostat. For detection of human LRRK2 protein, brain sections were incubated sequentially with a rabbit polyclonal antibody to the epitope HA (1:200; Rockland), with biotinylated goat anti-rabbit IgG (1:500; Vector Laboratories), and with peroxidase-conjugated avidin-biotin complex (ABC kit; Vector Laboratories). After thorough washing, bound antibodies were visualized by addition of diaminobenzidine (Vector Laboratories). Immunostained cells were observed under a Nikon microscope and were documented with a Nikon digital camera.

Rotarod test

Rats were first trained to stay on the rod of a rotarod (Med Associates) at a constant speed of 5 rpm, once a day for 5 days. Following the training, rats were tested on the Rotarod with an accelerating speed of 0.2 rpm/s, starting at 5 rpm. During a testing day, each rat was given three successive trials with a 20-minute interval. The latency to fall from the rod was recorded for each trial. The average latency was calculated for each testing day.

Statistical analysis

Data are expressed as means + SD. The statistical significance was tested with analysis of variance (ANOVA) followed by Tukey's post hoc test to compare group means. In all analyses, the null hypothesis was rejected at a level of 0.05.

Copy-related expression of the tTA transgene in CAG-tTA transgenic rats

Production of transgenic rats appears more difficult than creation of transgenic mice because the pronuclei of rat zygotes are less regular and less uniform than those of mouse zygotes. Furthermore, the plasma and pronuclear membranes of rat zygotes are more plastic and sticky than those of mouse zygotes. About 31-75% of rat zygotes are viable after microinjection and are ready for implantation into pseudopregnant females ^{2, 30}. We injected 540 single-cell embryos of SD rats with the CAGtTA transgene DNA at a concentration of 2ng/µl (microinjection buffer: Invitrogen) and transplanted 386 viable embryos into 12 pseudopregnant SD female rats (Figure 1A). From 68 pups, we identified three founder rats carrying the CAGtTA transgene (Figure 1B). Two of the three transgenic founders transmitted the transgene (Figure 1B: lines 5 and 11); however, founder 4 was infertile. We determined the copy number of the transgene in individual lines by quantitative PCR and detected 2 copies of the CAGtTA transgene in the line 5, 8 copies in the line 11, and 25 copies in the founder 4. The transgene was integrated at a single location in the genomes of the lines 5 and 11 because the copy number of the CAGtTA transgene was constant across generations. In addition, we tried to breed tTA transgenic lines to obtain homozygotes and succeeded with line 5 but not with line 11 (0 homozygotes in 30 transgenic offspring). No difference was observed in gross health between the homozygote and the hemizygote tTA rats.

To detect transgene expression, we first tried immunoblotting with commercially available antibodies that were raised with peptides corresponding to the tTA protein. Unfortunately, we could not detect tTA protein by western blotting in the two transmittable lines (Figure 1). In previous studies, low expression of tTA transgene has been shown sufficient to vigorously activate a tTA-dependent reporter gene ^{26, 39}. We designed a pair of primers spanning the intron of tTA transgene, and thus, we could identify mature tTA mRNA by RT-PCR (Figure 1A). We detected expression of the tTA transgene in various tissues of the two transmittable lines (Figure 1C). In many tissues, the tTA transgene was expressed at higher levels in the 8-copy line (line-11) than in the 2-copy line (line-5), suggesting a pattern of transgene copy-related expression.

Vigorous activation of tTA-dependent reporter gene in the double transgenic rats

To test whether limited levels of tTA gene expression were sufficient to activate its target genes in vivo, we crossed the tTA transgenic lines with a tTA-responsive reporter line that carried human LRRK2 gene under control of the TREminiCMV promoter (Figure 2A). By RT-PCR, we detected a widespread expression of the reporter gene in many tissues examined (Figure 2B). Since the reporter gene was tagged by hemaglutinin (HA), expression of the reporter gene could be detected by immunoblotting or immunohistochemistry for HA antigen. HA immunoreactivity was detected only when both the tTA and the reporter genes were presented in the transgenic rats, but was not detected when only the reporter transgene was present (Figure 2C). These results indicate that expression of the reporter gene depended on tTA activation and that leakage of the reporter gene was negligible. Further, western blotting detected strong HA immunoreactivity in the double transgenic rats carrying the 8-copy tTA transgene, but detected relatively weak HA immunoreactivity in the rats carrying 2-copy tTA transgene (Figure 2C). Expression levels of the reporter gene corresponded with those of the tTA transgene. In addition, immunohistochemistry with an HA antibody detected widespread expression of the reporter gene in many tissues of double-transgenic animals, but no expression in tissues of animals carrying the reporter transgene alone (Figure 2D-G). Consistent with the previous findings in transgenic mice ^{26, 39}, our results suggest that limited levels of tTA transgene expression were sufficient to vigorously activate its reporter gene in vivo.

Suppression of tTA-activated gene expression by Dox

One advantage of the tetracycline-regulatory system is that transgene expression can be suppressed temporally. To test whether the tTA-activated gene was responsive to Dox, we first treated adult rats carrying both the tTA and the reporter transgenes. When administered at a constant concentration in drinking water, Dox effectively suppressed expression of the tTA-dependent reporter gene and reached its maximal effect in a dose-dependent pattern (Figure 3A, B). By day 5, Dox administered at 500 μg/ml reduced mRNA and protein levels of the reporter gene below detection threshold. In contrast, lower doses of Dox took a longer time to reach its maximal effects: 100 μg/ml and 20 μg/ml of Dox reached the maximal effect by day 7 and by day 10, respectively. To improve drug efficiency, we tested a complex regime of Dox administration: Dox administered at 500 μg/ml for 2 days and then at 20 μg/ml for the remainder of the experiment. The complex (Cp) regime reached maximal effect by day 5 and achieved a comparable result to the highest dosage for Dox (Figure 3A, B).

To study the functions of embryo-lethal genes in adult animals, we should avoid expression of genes at the embryonic and postnatal stages. Therefore, we tested the gene-suppression efficiency of milk-secreted Dox in postnatal rats. Expression of the reporter gene was effectively suppressed in postnatal rats from 2 days of age through weaning when their mother was given Dox (20 μg/ml) in drinking water since the date of mating (Figure 3C, D). Our results show that expression of the tTA-dependent transgene was subject to a tight and constant suppression by Dox in both adult and postnatal rats.

Reversibility of tTA-dependent gene expression after Dox withdrawal

Compared to other gene regulatory systems, such as the Cre-lox system, a big advantage of the Tet regulatory system is that gene suppression can be released by Dox withdrawal. We first tested gene restoration in adult rats that were treated with Dox for 20 days. Expression of the reporter gene was efficiently restored in all the rats treated with three regimes of Dox (Figure 4A, B). Sixty days were required to remove the remnant effect of Dox administered at 500 μg/ml, but only 40 days were required to eliminate the remnant effect of Dox administered at 20 μg/ml (Figure 4A, B). Restoration time of gene expression is inversely relative to the dosage of Dox administered. We then tested recovery of gene expression in the rats inherently treated with Dox. We observed that gene recovery time depended on the age at Dox withdrawal. Periods of 40, 60, and 90 days were needed to recover gene expression in the rats depleted of Dox at ages of 15, 30, and 60 days, respectively (Figure 4C, D). Rats grow faster at early ages than at later ages (Figure 5A). Early withdrawal of Dox may require less time to remove the remnant effect of Dox due to a dilution effect from physical body expansion.

No effect of the tTA transgene on animal growth or motor function

In transgenic studies, gene insertion may cause unwanted phenotypes due to interruption of other genes at the insertion site. In addition, overexpression of the transcriptional factor tTA may cause side effects ³⁹. We examined tTA transgenic rats for overall health, but failed to observe any abnormal phenotypes. No differences were observed in the size of liver, heart, kidney, and brain between the tTA transgenic and the nontransgenic rats (data not shown). Compared to nontransgenic littermates, the tTA transgenic rats gained body weight at comparable speeds during postnatal development (Figure 5A). We monitored motor function using Rotarod test and observed no difference in psychomotor function between tTA transgenic and nontransgenic littermates (Figure 5B). Insertion and expression of the tTA transgene did not affect the growth or psychomotor function of rats.