Generation and characterization of a Tet-On (rtTA-M2) transgenic rat

Yi, Sheng; Lin, Che‐Chern; Yue, Junming; Sukhwani, Meena; Shuttleworth, Jennifer J.; Chu, Tianjiao; Orwig, Kyle E.

doi:10.1186/1471-213x-10-17

Cited by 14 publications

(9 citation statements)

References 49 publications

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“…Addition of doxycycline to the drinking water is a simple and effective method of drug administration 3, 26, 27, 37, 39 . However, there is inherent variability with this method because free water intake by individual animals is not well-controlled.…”

Section: Discussionmentioning

confidence: 99%

Effects of doxycycline on mesenchymal stem cell chondrogenesis and cartilage repair

Lee

O’Malley

Friel

et al. 2013

Osteoarthritis and Cartilage

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Objective Strategies to improve cartilage repair tissue quality after bone marrow cell-based procedures may reduce later development of osteoarthritis. Doxycycline is inexpensive, well-tolerated, and has been shown to reduce matrix-metalloproteinases (MMP) and osteoarthritis progression. This study tests the hypotheses that doxycycline reduces MMP, enhances chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSC), and improves in vivo cartilage repair. Design Ninety hMSC pellets were cultured in chondrogenic media with either 0-, 1- or 2-μg/mL doxycycline. Pellets were evaluated with stereomicroscopy, proteoglycan assay, qRT-PCR, and histology. Osteochondral defects (OCD) were created in the trochlear grooves of 24-Sprague-Dawley rats treated with/without oral doxycycline. Rats were sacrificed at 12-weeks and repair tissues were examined grossly and histologically. Results hMSC pellets with 1-μg/mL (p=0.014) and 2-μg/mL (p=0.002) doxycycline had larger areas than pellets without doxycycline. hMSC pellets with 2-μg/mL doxycycline showed reduced mmp-13 mRNA (p=0.010) and protein at 21-days. Proteoglycan, DNA contents, and mRNA expressions of chondrogenic genes were similar (p>0.05). For the in vivo study, while the histological scores were similar between the two groups (p=0.116), the gross scores of the OCD repair tissues in doxycycline-treated rats were higher at 12-weeks (p=0.017), reflective of improved repair quality. The doxycycline-treated repairs also showed lower MMP-13 protein (p=0.029). Conclusions This study shows that doxycycline improves hMSC chondrogenesis and decreases MMP-13 in pellet cultures and within rat OCDs. Doxycycline exerted no negative effect on multiple measures of chondrogenesis and cartilage repair. These data support potential use of doxycycline to improve cartilage repair to delay the onset of osteoarthritis.

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Section: Discussionmentioning

confidence: 99%

Effects of doxycycline on mesenchymal stem cell chondrogenesis and cartilage repair

Lee

O’Malley

Friel

et al. 2013

Osteoarthritis and Cartilage

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“…Doxycycline is generally administered through intraperitoneal injection, drinking water, or animal chow, and is systemically distributed to the entire body through the circulation. Therefore, doxycycline is accessible to almost all of the cell types in the body (3,(16)(17)(18)(19). Because most of the murine tTA and rtTA driver lines used to induce transgene expression rely on promoters that drive the expression of genes in multiple tissues, to achieve exclusive expression in an organ of interest with systemic doxycycline administration is often impossible.…”

mentioning

confidence: 99%

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

Tata

Pardo–Saganta²,

Prabhu³

et al. 2013

Am J Respir Cell Mol Biol

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Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter-driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell-specific transgene expression using a cytokeratin 5 (also known as keratin 5)-driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype.

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“…Cells were rinsed with phosphate-buffered saline (PBS), and fresh medium containing BSD was added with the same doses every other day. After 7 days of selection, TET-modified pTF promoter was induced by adding doxycycline (DOX) to a final concentration of 1 lg/ml, which was found to be the optimal concentration for rtTA2s-M2 in our constructs, as previously reported by others (Koponen et al, 2003;Sheng et al, 2010). Expression of the living color was analyzed by flow cytometry on a BD FACScan Flow Cytometer.…”

Section: Bsd Selection and Gene Expression Assaymentioning

confidence: 92%

Polyswitch Lentivectors: “All-in-One” Lentiviral Vectors for Drug-Inducible Gene Expression, Live Selection, and Recombination Cloning

Giry-Laterrière¹,

Cherpin²,

Kim

et al. 2011

Human Gene Therapy

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Lentiviral vectors are now widely considered one of the safest and most efficient tools for gene delivery and stable gene expression. Even though inducible gene expression cassettes are mandatory for many genetic engineering strategies, most current systems suffer from various issues, such as the requirement of two vectors, which decreases the overall efficiency of the transduction, leakiness and/or insufficient levels of activation of the inducible promoter, lack of selectable marker, low titers, or general issues associated with the cloning of large plasmids. In this article, we describe the design and functional characterization of a set of "all-in-one" multicistronic autoinducible lentivectors. They combine: (1) an optimized drug-inducible promoter; (2) a multicistronic strategy to express living color, selectable marker, and transactivator; and (3) acceptor sites for easy recombination cloning of genes of interest. These polyswitch lentivectors have good titers, very low basal activity, and reversible high induced activity, and can accept a growing number of genes already cloned in entry plasmids. These combined features make them a novel, powerful, and versatile tool for current and future genetic engineering approaches.

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Generation and characterization of a Tet-On (rtTA-M2) transgenic rat

Cited by 14 publications

References 49 publications

Effects of doxycycline on mesenchymal stem cell chondrogenesis and cartilage repair

Effects of doxycycline on mesenchymal stem cell chondrogenesis and cartilage repair

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

Polyswitch Lentivectors: “All-in-One” Lentiviral Vectors for Drug-Inducible Gene Expression, Live Selection, and Recombination Cloning

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