Biomedical subjects

Hermann Bujard

Publications and source records attributed to Hermann Bujard.

22 records · Page 2Linked to original sources

PubMed2002

Stringent doxycycline dependent control of CRE recombinase in vivo.

The strategy of modulating gene activities in vivo via CRE/loxP recombination would greatly profit from subjecting the recombination event to an independent and stringent temporal control. Here, we describe a transgenic mouse line, LC-1, where the expression of the cre and luciferase gene is tightly controlled by the Tet system. Using the R26R mouse line as indicator for CRE activity, and mouse lines expressing tetracycline controlled transactivators (tTA/rtTA) in various tissues, we show that; (i) in the non-induced state CRE recombinase is tightly controlled throughout the development and adulthood of an animal; (ii) upon induction, efficient recombination occurs in the adult animal in all tissues where tTA/rtTA is present, including hepatocytes, kidney cells, neurons and T lymphocytes; and (iii) no position effect appears to be caused by the LC-1 locus. Moreover, using the novel rTA(LAP)-1 mouse line, we show that in hepatocytes, complete deletion of the loxP-flanked insert in R26R animals is achieved less than 48 h after induction. Thus, the LC-1 mouse appears suitable for exploiting two rapidly increasing collections of mouse lines of which one provides tTA/rtTA in specific cell types/tissues, and the other a variety of loxP-flanked genes.

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PubMed2002

Studying gene function in eukaryotes by conditional gene inactivation.

The prospect of specifically controlling gene activities in vivo has become a defining hallmark of many model organisms of biological research. Where once the aim was to gain control over gene activities using endogenous control elements, new technologies have emerged that owe their remarkable specificity to heterologous components derived from evolutionarily distant species. This review highlights inducible transcriptional systems and site-specific recombination. Their quantitative and qualitative characteristics are discussed, with examples of how recent developments have expanded the spectrum of cells and organisms that are now accessible to genetic dissection of unprecedented precision. Transgenesis has already converted the mouse into a prime model for mammalian genetics. Combined with the new approaches of conditional activation or inactivation of genes, this model has opened up new horizons for the analysis of gene function in mammals.

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PubMed2002

Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators.

The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2(S)-S2 and rtTA2(S)-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2(S)-M2 and rtTA2(S)-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 microg of a single plasmid containing the rtTA2(S)-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2(S)-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

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PubMed2002

Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors.

We developed a panel of lentiviral vectors that displayed tetracycline-regulated transgene expression over two orders of magnitude in bulk, non-selected populations of transduced cells in vitro and in vivo. The robust expression and homogeneous response indicated that most transduced vector genomes were transcription competent and responsive to regulation, providing the lentiviral vector with a novel competitive advantage for gene transfer. After ex vivo transduction and transplantation of cord blood CD34+ cells into NOD/SCID mice, reporter gene expression could be switched "on" and "off" in human hematopoietic cells in vivo for prolonged times, proving integration of the regulated expression system into long-term repopulating cells. By vector injection into established tumor grafts, we achieved efficient delivery and quantitative regulation of transgene expression in vivo. By these approaches, gene function studies can now be performed in in vivo models of human hematopoiesis and cancer. In the future, regulated lentiviral vectors will improve the safety and efficacy of gene therapy.

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