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Construction and characterization of a doxycycline-inducible transgenic system in Msx2 expressing cells.

ABSTRACT: Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2-rtTA BAC transgenic line which can activate TetO-Cre expression in Msx2-expressing cells upon doxycycline (Dox) treatment. Using the Rosa26-LacZ (R26R) reporter line, we show that rtTA is activated in Msx2-expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, we show that in body appendages, the transgene can be activated in different domains depending on the timing of Dox treatment. In addition, the transgene can also be effectively activated in adult tissues such as the hair follicle and the urogenital system. Taken together, this Msx2-rtTA;TetO-Cre system is a valuable tool for studying gene function in the development of the aforementioned organs in a temporal and spatially-restricted manner, as well as for tissue lineage tracing of Msx2-expressing cells. When induced postnatally, this system can also be used to study gene function in adult tissues without compromising normal development and patterning.

SUBMITTER: Lin C

PROVIDER: S-EPMC2749712 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Construction and characterization of a doxycycline-inducible transgenic system in Msx2 expressing cells.

Lin Congxing C Yin Yan Y Chen Hong H Fisher Alexander V AV Chen Feng F Rauchman Michael M Ma Liang L

Genesis (New York, N.Y. : 2000) 20090501 5

Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2-rtTA BAC transgenic line which can activate TetO-Cre expression in Msx2-expressing cells upon doxycycline (Dox) treatment. Using the Rosa26-LacZ (R26R) reporter line, we show that rtTA is activated in Msx2-expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, ...[more]

PMID: 19370755

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Project description:PurposeIn the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.ProceduresIn this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.ResultsIn Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.ConclusionsOverall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Dataset Information

Construction and characterization of a doxycycline-inducible transgenic system in Msx2 expressing cells.

Publications

Construction and characterization of a doxycycline-inducible transgenic system in Msx2 expressing cells.

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