A versatile single-plasmid system for tissue-specific and inducible control of gene expression in transgenic mice

Abstract

We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.

Figure 1.

Tight control of reporter gene expression using the TRE-P_miniCMV system. A) pTL is the backbone plasmid that only contains rtTA and tTS cDNAs. TRE/loxP-inducible GFP (pTLiG) and TRE/loxP-inducible luciferase (pTLiL) are plasmids used to test the Dox-inducibility of reporter gene expression. In pTLcG, the GFP reporter gene is constitutively driven by the P_SV40 promoter. B) Examination of Dox-inducible GFP expression of pTLiG in HEK293 and CHO cells. HEK293 or CHO cells in 6-well plates were transfected with 1.0 μg of pTLiG or pTLcG plasmid. At 24 h after transfection, the cells were cultured further in a medium with or without Dox (1.0 μg/ml) for 24 h. GFP expression from transfected HEK293 or CHO cells was observed by fluorescence microscopy. C) Examination of Dox-inducible luciferase expression of pTLiL in HEK293 and CHO cells. HEK293 or CHO cells on 24-well plates were transfected with 0.5 μg of pTLiL or pTL plasmid. At 24 h after transfection, the cells were cultured further with or without Dox (1.0 μg/ml) for 24 h. Expression levels of luciferase were measured from HEK293 or CHO cells. Data show tight regulation of gene expression (<12% of leaky expression) in the absence of Dox, as compared with modified U6 promoters containing TRE sequence (see Supplemental Fig. S1) and robust induction in the presence of Dox (8- to 17-fold induction of luciferase reporter genes, depending on cell type).

Figure 2.

Single-plasmid-based system for inducible and tissue-specific control of gene silencing in transgenic mice. A) Schematic diagrams of pTLcG-mirJP system. Minimal CMV promoter P_miniCMV is linked to a TRE, a specific binding site for Tet-responsible transcription factors tTS and rtTA, which are expressed constitutively by a separate SV40 promoter. An internal ribosomal entry site (IRES) sequence permits translation of the 2 open reading frames for tTS and rtTA from single mRNA. A sequence of shRNA-mirJP targeting both JP1 and JP2 mRNAs is located downstream of an internal GFP expression cassette, which is flanked by 2 unidirectional loxP sites. B) Schematic of the experimental strategy in transgenic mice. First, the transgenic founder mice constitutively express tTS, rtTA, and GFP but will not express shRNA-mirJP in any cell. Transgenic offspring mice carrying both pTLcG-mirJP and muscle-specific Cre recombinase produce a functional Tet-on system for shRNA expression due to excision of GFP expression cassette by Cre-mediated recombination in a muscle-specific manner. Without Dox, the tTS molecules bind TRE and actively silence the transcription of the downstream shRNA-mirJP. In the presence of Dox, the shRNA-mirJP can be expressed actively by rtTA and processed by Drosha and Dicer to produce siRNA against JP1 and JP2 mRNA (inset).

Figure 3.

In vitro assay with Dox-induced knockdown of JP1 and JP2 expression using pTLcG-mirJP system. A) For testing the Dox-induced silencing of JP1 and JP2 expression, the GFP expression cassette was excised from the pTLcG-mirJP plasmid to generate the pTLi-mirJP construct (which mimics Cre-recombination of the pTLcG-mirJP system). B) HEK293 cells in 6-well plates were cotransfected with 1.0 μg of pTLcG-mirJP or pTLi-mirJP together with 0.2 μg of either pCMS-EGFP-mJP1 or pCMS-RFP-mJP2 (41). Dox (1.0 μg/ml) was applied 24 h after transfection. Total cell lysates were prepared at 24 h after addition of Dox, and 10 μg of proteins was analyzed by Western blotting against mouse JP1 or JP2. C) HEK293 cells were cotransfected with 1.0 μg of pTLi-mirJP and 0.2 μg of pCMS-EGFP-mJP1 or pCMS-RFP-mJP2. At 24 h after transfection, cells were treated with indicated concentrations of Dox. Cells were cultured further for 24 h and assayed by Western blotting. D) HEK293 cells cotransfected with 1.0 μg of pTLi-mirJP and 0.2 μg of pCMS-EGFP-mJP1 were treated with Dox (1.0 μg/ml) at 24 h after transfection. After 48 h incubation in the presence of Dox, cells were washed and incubated further in fresh medium as indicated. Expression of JP1 was detected by Western blotting. E) To analyze in vitro Cre recombination, 200 ng of pTLcG-mirJP was incubated with Cre enzyme (1 U) at 37°C for 30 min, followed by heat inactivation of Cre at 70°C for 10 min. Plasmids were purified from the reaction mixture, and the recombined plasmids were analyzed by PCR, using 2 primers flanking the GFP expression cassette. Open arrow indicates unrecombined product (2030 bp) containing the GFP expression cassette; solid arrow indicates recombined product (470 bp). F) HEK293 cells were transfected with 0.2 μg of pTLcG-mirJP with or without 1.0 μg of pTurbo-Cre. From 24 h after transfection, cells were observed by fluorescence microscopy to detect GFP expression.

Figure 4.

Quantitative assay of Dox-inducibility and reversibility of luciferase reporter activity using the pTLcG system. A) For a quantitative analysis of the system, shRNA-mirJP sequence in the pTLcG-mirJP and pTLi-mirJP was replaced with shRNA-mirLuc sequence targeting fLuc mRNA to construct pTLcG-mirLuc and pTLi-mirLuc. B) CHO or HEK293 cells in 24-well plates were cotransfected with 0.5 μg of pTLcG-mirLuc or pTLi-mirLuc and 0.1 μg of pDualuc. At 24 h after transfection, cells were treated with or without Dox (1.0 μg/ml) and incubated further for 24 h. Dual luciferase assays were carried out in triplicate; results represent ratio of Renilla to firefly luciferase activity (mean±se). C) HEK293 cells in 24-well plates were cotransfected with 0.5 μg of pTLcG-mirLuc or pTLi-mirLuc and 0.1 μg of pDualuc. At 24 h after transfection, cells were treated with indicated concentrations of Dox. Cells were cultured further for 24 h and analyzed by dual luciferase assay. D) HEK293 cells cotransfected with pTLi-mirLuc and pDualuc were treated with Dox (1.0 μg/ml) at 24 h after transfection. After 48 h incubation in the presence of Dox, cells were washed and further incubated in Dox-free medium. At indicated time points, cells were prepared and analyzed by dual luciferase assay.

Figure 5.

Muscle-specific knockdown of JP1 and JP2 expression using the mirJP/Cre transgenic mice. pTLcG-mirJP transgenic (mirJP) mice were mated with HSA-Cre79 transgenic (cre) mice to generate offspring carrying both transgenes (mirJP/Cre). Eight-week-old F2-mirJP/cre mice were examined to detect Cre-mediated DNA recombination (A, B) and Dox-dependent JP knockdown (C, D). A) Genomic PCR was performed in various tissues of wild-type (wt), pTLcG-mirJP (mirJP), and pTLcG-mirJP/HSA-cre (mirJP/cre) transgenic mice. Cre-mediated recombination was determined by detection of two different sizes of PCR products, 2030 bp for unrecombined and 470 bp for recombined products. M, 100-bp DNA ladder marker; Sk, skeletal muscle; Ht, heart; Lu, lung; Li, liver; Sp, spleen; Kd, kidney. Asterisks indicate nonspecific PCR products. B) Gastrocnemious muscles prepared from the mice were cross-sectioned (5 μm thickness) and examined under fluorescence microscopy to visualize the expression pattern of GFP. Area enclosed by dashed line corresponds to fibers showing negative or very weak GFP fluorescence on the section. GFP-positive fibers were counted from the same sections (right panel). C) Examination of Dox-dependent reduction of the JP expression in skeletal muscle. Eight-week-old wt and mirJP/cre mice were supplied with water containing 0.2 or 2.0 mg/ml of Dox for 1 wk. Gastrocnemious muscles were isolated, and expression levels of JP1 and JP2 were examined by Western blotting. D) Reversible control of Dox-inducible knockdown of JPs in skeletal muscle. Eight-week-old wt and transgenic mice were provided with water either with or without Dox (2 mg/ml) for 2 wk. One cohort of Dox-treated mirJP/cre mice was sacrificed (Dox + group), and the other cohort was supplied with Dox-free water for 4 wk to inactivate Tet-on system for shRNA-mirJP expression and recover expression level of JP1 and JP2 (Dox +→− group). Gastrocnemious and cardiac muscles were prepared and examined by Western blotting for JP-1 and JP-2. Each lane shows Western blotting for the muscle isolated from different mice.

Figure 6.

Inducible knockdown of JPs leads to abnormal triad junction ultrastructure in transgenic skeletal muscles. EM analysis was performed on EDL muscles isolated from 3–7 mice/experimental group. A–C) Representative electron micrographs of muscle tissue from Dox-untreated mirJP/cre mice (A), Dox-treated mirJP/cre mice (B), and water-switched mirJP/cre mice after Dox-treatment (C). Arrows indicate irregular triad junctions. Top panels: lower-magnification EM images of longitudinal EDL section. Bottom panels: representative high-magnification images of triad junctions. D) Statistical analysis of changes in junctional membrane structures. At least 400 junctional membrane structures from each EDL muscle fiber were examined. Data are presented as means ± se. Scale bars = 1 μm (top panels); 0.2 μm (bottom panels).

Figure 7.

Knockdown of JPs decreases thapsigargin-induced Ca²⁺-release from SR and SOCE in transgenic skeletal muscle fibers. To measure Ca²⁺ release from SR and SOCE, FDB muscle fibers were isolated and loaded with 10 μM Fura-2 AM. SR Ca²⁺ stores of fibers were depleted by the addition of 20 μM TG (7 min). Addition of 0.5 mM Mn²⁺ (5 min) leads to quenching of Fura-2 fluorescence at excitation wavelength of 360 nm (F₃₆₀), indicating activation of TG-induced SOCE in FDB fibers. During the experiments, basal value of F₃₆₀/F₃₉₀, indicating resting cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt), was measured before the addition of TG, and the change of F₃₆₀/F₃₉₀ ratio (ΔF₃₆₀/F₃₉₀), indicating TG-induced Ca²⁺ release from SR, was measured after the addition of TG. A) Average data for the basal value of F₃₆₀/F₃₉₀. Result shows no significant difference among groups. B) Average data for the TG-induced change of F₃₆₀/F₃₉₀. C) Representative traces of F₃₆₀, measured from FDB fibers of Dox-untreated wt mice (a), Dox-treated wt mice (b), Dox-untreated mirJP/cre mice (c), Dox-treated mirJP/cre mice (d, e), and water-switched mirJP/cre mice after Dox-treatment (f). Fura-2 fluorescence is normalized to a maximum of 1.0 and a minimum of 0 for the value after permeabilization with Triton X-100. D) Statistical analysis of multiple SOCE measurements. Light gray circles indicate rate of Mn²⁺ quenching of Fura-2 fluorescence from individual fibers; dark gray circles indicate data from F₃₆₀ traces in C. Horizontal bar indicates mean values in each group. Experiments were performed on the fibers (n=8–22) from ≥3 mice/group. NS, not significant (P>0.05. Note that some fibers with Dox treatment show apparently normal values of SOCE compared with the untreated fibers, which likely reflects the incomplete Cre-recombination system in the muscle background. E) In vitro fatigability and force recovery profile of control (left panel) and JP-KD (right panel) EDL muscle bundles. Traces are representative of 6 muscle bundles in the control group; 4 muscle bundles in the JP-KD group.