Tetracycline-Regulated Gene Expression in Transgenic Mouse Epidermis
The ability to specifically manipulate gene expression in vivo using mouse models has been one of the most important advances in understanding gene function over the last few decades. Methods used to control gene activities in the mouse include gene targeting and transgenic approaches. While gene targeting methods have proven to be powerful genetic tools designed to eliminate gene function by creating a “knockout” or “null mutant,” transgenic studies offer gain-of-function capabilities, expression of dominant negative or knockdown of specific target genes and thus have often served a complementary and useful role. This chapter provides an overview for the generation of transgenic mouse models to study important questions in skin biology by taking advantage of the tetracycline-inducible gene expression system.
Key wordsKeratinocyte Transgenic Tet-Effector Tet-Responder Skin Inducible
It is relatively easy (with practice and skill!) to insert a foreign piece of DNA, i.e., a transgene, into the mouse genome to generate a transgenic mouse. For this purpose the foreign DNA is introduced using a fine needle directly into the mouse egg, where the DNA integrates at a random position in the genome – a procedure referred to as “pronuclear injection.” This typically results in the incorporation of many tandemly arranged copies of the transgene in the genome and thus generates animals with increased levels of expression of the gene or gene product of interest. In addition, this system can also be used to generate dominant negatives or deliver siRNA to inhibit the expression pattern of a specific endogenous gene by using cell- and tissue-specific promoters. However, one of the major shortcomings of this system is that constitutive expression of the transgene can sometimes have toxic and/or deleterious effects on the animal resulting in embryonic or early postnatal lethality. Furthermore, this technique does not allow for the manipulation of transgene expression in a spatially or temporally defined manner or to alter the desired expression levels of the transgene. These drawbacks can make studying in vivo gene function rather challenging.
The second component consists of animals expressing an inducible transgene of interest under the control of a tetracycline-regulated promoter or operator (tetO) (also known as a tetracycline-responsive element, or TRE). In this case, binding of the TetR to the TRE regulates gene expression. Thus, the tetracycline-inducible system is a binary system in that it requires the generation of two separate transgenic lines of mice: one carrying the tTA or rtTA transgene (Tet-Effector mice) and another carrying the transgene of interest driven by the TRE (Tet-Responder mice). These transgenic animals can then be cross-bred to produce bi-transgenic animals and transgene expression can be tightly regulated by the administration or withdrawal of Dox (seeFig.20.1). For example, in the previously described Tet-On system, bi-transgenic animals can be administered Dox through their diet or in their drinking water, allowing transcriptional activation of the transgene. Upon the withdrawal of Dox, transgene expression is abolished. Conversely, in the Tet-Off system, in the absence of Dox, transgene expression is induced.
One of the major benefits of the Tet-inducible system is that it allows for varied expression levels of the transgene and provides the option to rapidly and reversibly switch the transgene on or off at any time point during the lifetime of the animal. Stringent control over transgene expression can be achieved by changing the concentrations of Dox that are administered to the bi-transgenic animals. Another advantage is the highly effective tissue- and cell-penetrant properties of Dox; it is transferred across the placenta and through the breast milk of lactating mothers. These factors make this system ideal for inducing transgene expression during embryonic development as well as in the offsprings before weaning (3). Finally the expression of the TetR can be driven by a cell- or tissue-specific promoter allowing restricted and targeted expression of the transgene, thereby avoiding systemic effects. The versatility of this system is further enhanced by the fact that a growing number of Tet-Effector transgenic animals are available commercially and from different research laboratories.
2.1 Plasmids for Gene Cloning
2.1.1 TRE Plasmids
pTRE-HA,-Myc plasmids contain a TRE, a CMV minimal promoter, and an N-terminal epitope tag as indicated, followed by a MCS (Clontech Cat. no. 631012 and 631010, respectively).
pTRE-Tight vectors contain a modified TRE and minimal CMV promoter that provide better control of gene expression by eliminating leaky transgene expression in the absence of inducer. However, this plasmid lacks both an epitope tag and an initiating ATG, both of which can be easily incorporated by routine molecular biology techniques (Clontech Cat. no. 63159).
2.2 Keratinocyte-Specific Tet-Effector Transgenic Animals
A more comprehensive database of Tet-Effector and Tet-Responder transgenic animals is available at the following site http://www.zmg.uni-mainz.de/tetmouse.
2.3 Other Key Reagents
Doxycycline (Sigma-Aldrich, St,Louis, MO; Cat. no. D9891) is a tetracycline derivative commonly used by most laboratories. For long-term storage, prepare a sterile 1 mg/ml stock in sterile water, filter sterilize, aliquot, and store at –20°C. The final concentration used in a typical cell culture experiment is typically 0.2–1 μg/ml of culture medium.
OmniPrep Kit for Extraction of High-Quality Genomic DNA (GBiosciences Cat. no. 786-136). This kit can be used for extracting genomic DNA from animal samples quickly and with relative ease.
Histo-ClearII (National Diagnostics Cat. no. HsS-202). This is used for de-paraffinization of slides which have paraffin-embedded tissue samples on them.
QIAfilter Plasmid Midi Kit (Qiagen, Cat. no. 12243).
QIAquick Gel Extraction Kit (Qiagen, Cat. no. 28704).
Tet system approved Fetal Bovine Serum (Clontech, Cat. no. 631106).
2.4 Genomic DNA Extraction Buffer
Proteinase K stock solution at 20 mg/ml (Novagen Cat. no. 70663-4). Dissolve in 10 mM Tris HCL, pH 7.5, 20 mM calcium chloride, and 50% glycerol. Store in 0.5 ml aliquots at –20°C.
DNA Extraction Buffer:50 mM Tris (pH 8.0), 10 mM EDTA, 200 mM NaCl, 0.5% SDS.
2.5 Mouse Dissections and Skin Tissue Specimen Preparation
Following the animals care guidelines set out by the Institutional Animal Care and Use Committee (IACUC), both a wild-type and bi-transgenic mouse must be sacrificed.
Using an electric shaver, gently shave the area of the skin that needs to be harvested (typically the dorsal region of the mouse). Working quickly to minimize tissue damage and necrosis, harvest the skin. Spread the skin out flat with the dermis side down on a paper towel. Trim the excess paper towel and cut into a small-sized piece and proceed directly to fixation.
Tissues can be fixed in 10% Neutral Buffered Formalin (NBF) overnight at room temperature (RT). The tissues are then processed and embedded in paraffin and sectioned to 4–10 μm thickness. Tissues can alternatively be fixed in 4% paraformaldehyde overnight at RT.
2.6 Blocking Solutions
For 50 ml: NGS (10 ml), 10% TX-100 (0.5 ml),10× PBS (5 ml), MilliQ water (34 ml), 2% NaN3 (0.5 ml), make the solution first, then heat inactivate at 56°C for 30 min. Store at 4°C.
2% NGS in 0.1% TX-100 in PBS
For 10 ml: Blocking solution (see above) (1 ml), 10% TX-100 (90 μl), 10× PBS (900 μl),
MilliQ water (7.92 ml), 2% NaN3 (90 μl). Store at 4°C.
3.1 Cloning the Transgene and Preparing the Plasmid for Microinjection
3.1.1 Plasmid Design
When cloning the transgene into a TRE plasmid containing an epitope tag, the coding sequence of the gene must be placed in frame with the epitope tag. This can be done using simple PCR strategies. It is important to then sequence the cloned transgene for errors, which may have occurred during the PCR, and to ensure the transgene is in frame with the epitope tag.
Once the transgene sequence has been verified, Tet-regulated expression of the designed transgene can be tested in cell culture by Western Blot analysis. Tet-On and Tet-Off plasmids are available from Clontech and can be used in co-transfection experiments with the TRE plasmid containing your transgene. This is a critical preliminary step whose importance cannot be stressed enough. This ensures that the transgene is expressed as expected and thus can save valuable time and resources associated with the generation of transgenic mice.
3.1.2 Preparing the Plasmid for Microinjection
Once Tet-regulated expression of the transgene has been confirmed, the plasmid can be prepared for microinjection by excising the transgene from the plasmid backbone using appropriate restriction enzymes and gel electrophoresis. Linearization of the transgene has been shown to improve integration efficiency and removal of the extraneous plasmid sequences can improve the frequency of expression (13). When considering removal of the plasmid backbone, be sure not to remove the polyadenylation signal within the backbone, as this will prevent transgene expression in vivo (seeFig.20.2). Techniques and protocols for the purification of the plasmid for microinjection should follow the guidelines set out by the transgenic facility performing the microinjection. There are several factors that may influence the success rate of generating transgenic animals with the most important being the purity and the quality of the DNA used for microinjection (seeNote 1). DNA concentration is another factor influencing the success rate of generating transgenic animals.
Injection of the DNA-containing solution into the pronuclei of fertilized eggs is the most common and efficient method used for generating transgenic animals. With this approach, the transgene undergoes random integration into the mouse genome, often allowing multiple transgene copies to be inserted at a single locus in a head-to-tail fashion. There are a number of shortcomings using this technique in that due to the random nature of insertion of the transgene within the mouse genome, it is possible this method can result in potential integration into sites of permanently silenced chromatin. In this case, the transgene will not express. Another possibility to consider is the potential for mosaicism. Although the transgene is generally transmitted in a Mendelian fashion, in the case of a mosaic mouse, transmission may not follow Mendelian frequencies, and one may have to go though several breeding cycles to generate an offspring carrying the transgene. As a result, it is wise to work with multiple (preferably three to five or more) different founder lines to ensure an expressing animal and to confirm the observed phenotype.
3.2 Screening and Identification of Founder Transgenic Lines
Take a 1–2 mm tail biopsy from a 2-week-old mouse.
Extract genomic DNA: Add 250 µl of DNA extraction buffer and 3.5 µl of proteinase K solution (20 mg/ml) to the tail biopsy and incubate at 55°C overnight. Add an equal volume of phenol/chloroform (1:1), mix well and centrifuge at 15,000g for 5 min at room temperature (RT). Remove the supernatant and place in new centrifuge tube and add 500 µl of 95% ethanol. Centrifuge for 5 min at 15,000g at RT. Wash the DNA pellet with 70% ethanol, decant the pellet, and let air dry. The pellet can be dissolved in 100 µl of ddH2O and stored at 4°C for several weeks. For PCR reactions, 1 μl of genomic DNA can be used.
Conversely, the OmniPrep Kit for Extraction of High Quality Genomic DNA available from GBiosciences can also be used. See materials section above.
3.2.1 Genotyping Potential Founders Using PCR
Once genomic DNA has been isolated, PCR can be performed to identify transgenic founders. Although the quality of genomic DNA is a contributing factor in the identification of false positives or false negatives, an equally important factor is the primer sets used for genotyping. When designing primers for use in PCR genotyping reactions, it is important to use a set of primers that will amplify a region within the transgene. Potential regions of amplification include the polyadenylation signal, or sequences spanning the MCS and the 5′ region of the gene. A reason these areas are ideal for primer design is that they generally lack any homology to the mouse genome and are less likely to give non-specific amplification. Given this, primers should not be designed within the coding region of the transgene as in some cases, this may not distinguish between the endogenous genomic DNA and the transgene.
3.3 Confirming Expression of the Transgene In Vitro
The identification of founder transgenic lines does not guarantee successful transgene expression in vivo. This depends on many factors, some of which have been addressed in the above sections. Although the most straightforward method to confirm expression of the transgene is to cross-breed the Tet-Responder transgenic founders to a Tet-Effector transgenic line, this may not be feasible if many founders have been identified. A simpler and faster approach to screen through a high number of transgenic founder lines takes advantage of the relative ease with which keratinocytes and fibroblasts can be isolated from mouse skin and maintained in culture. Fibroblast cell lines from transgenic founders can be generated from tails and subsequently tested for transgene induction by transfecting the cells with a TetR plasmid driven by the ubiquitously expressed CMV promoter (4). Transgene expression can be confirmed by performing a Western Blot to detect expression of the epitope tag. If multiple expressing founders have been identified, relative expression levels of the transgene can be extrapolated from Western blots and matings can be set up accordingly. Another advantage of utilizing the cell culture method for confirming transgene expression is that it can be used for monitoring the inherent leakiness of this system. One of the limitations of the Tet-inducible system is the high basal activity of the TRE promoter in uninduced states, resulting in leaky expression of the transgene. These conditions are generally unfavorable, particularly if the transgene is highly toxic. Transgenic founder lines can be screened for levels of leakiness by examining transgene expression levels in the absence of inducer (14).
3.4 Confirming Expression of the Transgene In Vivo
Once expressing founders have been identified, they can then be cross-bred to Tet-Effector transgenic animals expressing a TetR under the control of the keratinocyte-specific promoter of your choice. When choosing Tet-Effector animals to be used for cross-breeding, important factors to consider are some of the inherent limitations of both the rtTA and the tTA systems. While the tTA or Tet-Off system is very convenient in that in the absence of Dox, the transgene is induced, if the transgene requires a period of silencing followed by induction, depending on the Dox dosage, induction can take up to a number of days, and reaching steady-state levels can take several more days (5). This is because the circulating levels of Dox in the system require some time for elimination. However, this system does provide tight control of gene expression, thereby minimizing the likelihood of leaky transgene expression.
Conversely, the rtTA or Tet-On system is more suitable for rapid induction of transgene expression upon Dox administration. Depending on the organ or tissue, transgene induction can be detected within 4 hours when Dox is supplied in the drinking water of bi-transgenic animals (15). This is particularly useful in cases when overexpression of the transgene is toxic to the animal. A drawback of the Tet-On system is that the commonly used Tet transactivator utilized in this system has a lower affinity for Dox; however, recent modifications have enhanced its sensitivity to Dox and diminished residual binding to TRE in the absence of Dox, thus improving the inherent leakiness (16).
Once Tet-Effector animals have been mated to the newly generated Tet-Responder mice to generate bi-transgenic animals, depending on the Tet-inducible system being employed (Tet-On or Tet-Off), Dox should be administered accordingly. An important factor to consider when setting up matings and inducing transgene expression in the bi-transgenic animals is the experimental window under investigation. For example, to initiate expression during embryonic development and prior to weaning, Dox can be administered to the mother (for the Tet-on system). On the other hand, if using tTA Effector mice, Dox must be withdrawn to induce transgene expression. Another consideration to keep in mind is the nature of the keratinocyte-specific promoters driving the TetR, since their activity during embryonic skin development is variable and this will determine when the transgene is expressed (seeNote 2).
Deparaffinize the sections by gently shaking the slides in a glass coplin jar using histoclear for 3 min at RT. This can be repeated twice. Sections can then be rehydrated through a graded alcohol series. Gently shaking the slides in the coplin jar, add 95% alcohol for 3 min, followed by 70% alcohol for 3 min, and then 50% alcohol for 3 min. Slides can then be washed on the shaker in 1× PBS for a total of three times at 5 min each.
Antigen retrieval solution:
10 mM sodium citrate buffer (pH 6.0) containing 0.05% Tween-20.
The solution can be stored at room temperature (RT).
Place the deparaffinized slides in the antigen retrieval solution with the sections facing up in a glass pyrex microwave safe dish. Cover the pyrex dish with Saran Wrap and poke tiny holes in the Saran Wrap to allow for ventilation. Microwave at maximum power for 20 min, then cool for 20 min at RT after removing the Saran Wrap.
Arrange the cooled slides in a coplin jar and rinse three times with 1× PBS.
Suck off remaining PBS from the slides and circle the sections with a PAP pen, and place slides in a humidifying chamber.
Block using 20% Normal Goat Serum (NGS) with 0.1% Triton X-100 in PBS (blocking solution) for 60 min (or longer) at RT.
Add primary antibody by diluting the antibody in the 2% NGS. Incubate 2 hours (RT) or overnight at 4°C. Overnight is recommended if the transgene is weakly expressed.
Transfer slides to coplin jar and wash with 1× PBS three times for 5 min each on a shaker.
Place slides back in the chamber and add secondary antibody diluted in 2% NGS. Incubate at RT for 45 min.
Wash the slides three times with 1× PBS for 5 min on a shaker.
Rinse briefly in tap water and mount slides using Vectashield mounting medium for fluorescence with DAPI (cat# H-1200 Vector Laboratory).
Alternatively, slides can be blocked using 5% Bovine Serum Albumin (BSA) or 5% non-fat dry milk solutions.
3.5 Methods for Administering Doxycycline
Although there are several modes for administration of Dox for use in transgenic animals, the most commonly used method for delivery of Dox is through drinking water. An important parameter to consider is the amount of Dox required for effective transgene induction (seeNote 3). Dox dosage is variable in that different promoter-specific Tet-Effector transgenic lines may require different amounts of Dox for induction of the transgene. Dosage levels should be consistent with the levels suggested by the research group who initially generated and characterized the Tet-Effector transgenic line (typically 1.125 mg/ml to 10 mg/ml). This information should be widely available.
In addition to Dox delivery in drinking water, Dox can also be administered through rodent chow (Bio-Serv; Frenchtown, NJ). Varying concentrations are available and should be consistent with the recommended dose corresponding to the Tet-Effector transgenic animals you are using for your cross-breeding. The only drawback to using Dox chow is that if multiple Tet-Effector animals will be used, they may each require different amounts of Dox dosage. In this case, administering Dox through drinking water may be more sensible (seeNote 4 for important considerations).
Finally, the third most widely used alternative method of Dox delivery is through intraperitoneal (IP) injections. The main advantage of administering Dox using this method is that delivery and induction of the transgene are rapid. This form of delivery can be particularly useful in cases when the transgene is toxic to the animal. This mode of administration can provide transgene induction within 3 hours after delivery (10). Similar to the noted benefits of using Dox in drinking water, utilizing IP injections allows for increased variability in Dox dosage.
3.6 Mouse Strain Considerations
FVB mice are a commonly utilized strain of mice for the generation of transgenic animals due to their fully inbred genetic background. Use of this strain will ensure all founders to be genetically identical. This strain also provides reproductive advantages in that fertilized eggs contain large pronuclei facilitating the microinjection of DNA. In addition, FVB animals are highly fecund, generating large numbers of eggs and litters. These factors make this strain very efficient for making transgenics (18).
C57BL/6 is the most widely used inbred strain of mice. This strain has a good breeding performance, has a long life span, and is the most commonly used strain in biomedical research.
A second important factor to consider prior to microinjection is the mouse strain that will be utilized for cross-breeding in the future and whether the progeny will be analyzed on a pure or mixed genetic background. It is widely accepted that phenotypic differences between mouse genetic strains can influence the severity of the observed phenotype. For example, if your newly generated Tet-Responder transgenic animals will be mated to a pure FVB strain of Tet-Effector animals, you may want to keep the offspring on a pure FVB strain and may choose to use FVB donor eggs for microinjection. Maintaining the progeny on a pure background will reduce any observed variability. Conversely, if the progeny are bred on a mixed genetic strain, phenotypic variability between littermates is possible. However, one of the advantages of analyzing mice on a mixed strain is that the observed phenotype is more likely to represent the common phenotype across different genetic strains (19). If needed, transgenic mice on mixed backgrounds can be easily bred to generate a pure congenic strain by backcrossing for eight generations or more.
Successful generation of transgenic animals is dependent on a number of different variables including the purity and quality of the DNA. When purifying DNA for microinjection, keep in mind that CsCl preparations (preps.) are routinely suggested by many core facilities, which perform the transgene injections. In some cases, two rounds of CsCl preps. are requested. This is to ensure the highest quality and purity of DNA as the slightest traces of contaminating agents will affect successful generation of transgenic animals. If CsCl preps. are not feasible, we have found that DNA purification using the QIAfilter Plasmid Midi Kit works very effectively and provide highly pure DNA. If possible, endotoxin-free DNA is even a better option. After restriction digestion to eliminate the unnecessary plasmid backbone and gel electrophoresis, the excised band that will be injected can be purified using the QIAquick Gel Extraction Kit.
When planning experimental strategies one thing to consider is the relevant biological question and what cellular layer of the epidermis will be targeted for transgene expression. This will dictate which Tet-Effector animals will be utilized for mating to the newly generated Tet-Responder animals. A second parameter to consider is when to induce the transgene. If studying gene function during embryonic skin morphogenesis, then induction should occur during this developmental window. Conversely, if studying transgene function in adult skin, induction should occur in adult animals. For example, depending on the gene driving expression of the TetR, induction can occur as early as E8.5 (as seen with the K5/14 Tet-Effector animals) to as late as E16.5 (with the Involucrin Tet-Effector animals).
Optimal doxycycline dosage levels required to achieve maximal transgene expression vary between Tet-Effector transgenic mouse lines. Given this variability, it is wise to refer to the original report describing the initial characterization of the Tet-Effector animals. Generally, Dox dosage levels can range from 0.125 mg/ml to 10 mg/ml. When deciding on the dosage levels, it is important to consider the main goal of the research and the need to express the transgene at low or high levels. For example, in the case of a Tet-On system which is being controlled through Dox in drinking water, once animals are switched to regular water, animals kept on a low Dox dosage show a more significant decline of transgene expression as compared to animals on higher Dox dosages (12).
If administering Doxycycline to animals through drinking water, it is important to remember that Dox is light sensitive and requires storage in specially designed light-sensitive bottles when placed in the cages of mice. Alternatively, drinking bottles can be wrapped in aluminum foil to avoid exposure to light. However, this requires caution as mice can easily tear and claw at the foil and ingest it. In addition, the Dox drinking solution should be supplemented with 5% sucrose or Koolaid since Dox is not palatable and has a very bitter taste. If the solution is not supplemented and replenished appropriately, the animals might die of dehydration as reported in one study (20). Furthermore, to ensure the right concentration of Dox, the drinking solution needs to be made fresh every 2–3 days.
Mouse matings between Tet-Effector and Tet-Responder animals to generate bi-transgenic offspring should follow normal Mendelian frequencies. As basic as this may sound, careful attention should be paid to keep track of this as if you fail to identify bi-transgenic animals after a few litters, it may be due to the early postnatal lethality associated with expression of your transgene. This is particularly true if transgene expression is induced during the early stages of embryonic development. Given the fact that many of the promoters driving expression of the TetR in the skin are not restricted to the skin but are also expressed in various other stratified epithelial tissues such as the stomach and oral cavity, it is plausible that overexpression of the transgene in other tissues may be lethal. In such cases, induction during later stages of development, or at birth, or controlling the dosage of Dox may be required to circumvent this early lethality.
If you have positively identified a large number of transgenic founder lines and choose to distinguish the transgenic founder lines that express the transgene in a Tet-inducible fashion by isolating keratinocytes or fibroblasts, be sure to utilize cell culture reagents such as Fetal Bovine Serum (FBS) that are Tet-system approved and are free of tetracycline-derived contaminants.
To reduce the numbers of animals and minimize the breeding requirements that are associated with the bi-transgenic system, strategies that use co-injection or an integrated plasmid that harbors both components of the Tet-System can be utilized. For example, if plasmids bearing the Tet-Responder and the Tet-Effector units are co-injected into single-cell fertilized embryos, they typically co-integrate into the same site(s) of the genome. This will generate a single transgenic mouse that will respond to Dox. Alternatively, integrating all of the elements of the Dox-inducible system on a single “all-in-one” vector is another way to avoid generating binary Dox-responsive transgenic animal model systems. Although these strategies eliminate the need to generate two independent transgenic lines and greatly facilitates mouse breeding, an inherent shortcoming with this strategy is the fact that the scope of inducible transgene expression is then limited to the specific Tet-Effector of choice and cannot be utilized for a different organ or tissue system if desired.
Work in our laboratory is supported by National Institutes of Health Grant R01GM069417 and RO1AR049238 (S.S.).
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