Jost Seibler

The Transgeneticists Toolbox: Novel Methods for the Targeted Modification of Eukaryotic Genomes

Abstract Classical techniques for gene transfer into mammalian cells involve tedious screening procedures to identify transgenic clones or animals with the appropriate level and stability of expression or with the correct developmental patterns. These first generation technologies are clearly inadequate for complex genetic strategies by which gene regulation can be studied in its entire complexity. While site-specific insertions can principally be achieved by homologous recombination or by adapting the recombination apparatus from phages or yeast, these methods usually lack the required efficiency or they perturb expression patterns by the co-insertion of prokaryotic vector parts. Virtually all of these problems can be overcome by recombinase-mediated cassette exchange (RMCE) techniques which cleanly replace a resident cassette that is flanked by two hetero-specific recombination target sites for a second cassette with the analogous design, presented on a targeting vector. After illustrating the fundamentals of site-specific recombination by selected experiments, the authors (arranged in the chronological order of their contribution) will describe their efforts to develop RMCE into a method of wide applicability. Further developments that have been initiated utilizing the particular potential of the RMCE principle will be outlined.

Performance of Genomic Bordering Elements at Predefined Genomic Loci

ABSTRACT Eukaryotic DNA is organized into chromatin domains that regulate gene expression and chromosome behavior. Insulators and/or scaffold-matrix attachment regions (S/MARs) mark the boundaries of these chromatin domains where they delimit enhancing and silencing effects from the outside. By recombinase-mediated cassette exchange (RMCE), we were able to compare these two types of bordering elements at a number of predefined genomic loci. Flanking an expression vector with either S/MARs or two copies of the non-S/MAR chicken hypersensitive site 4 insulator demonstrates that while these borders confer related expression characteristics at most loci, their effect on chromatin organization is clearly distinct. Our results suggest that the activity of bordering elements is most pronounced for the abundant class of loci with a low but negligible expression potential in the case of highly expressed sites. By the RMCE procedure, we demonstrate that expression parameters are not due to a potential targeting action of bordering elements, in the sense that a linked transgene is directed into a special class of loci. Instead, we can relate the observed transcriptional augmentation phenomena to their function as genomic insulators.

Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice

The function of the transient receptor potential vanilloid 1 (TRPV1) cation channel was analyzed with RNA interference technologies and compared to TRPV1 knockout mice. Expression of shRNAs targeting TRPV1 in transgenic (tg) mice was proven by RNase protection assays, and TRPV1 downregulation was confirmed by reduced expression of TRPV1 mRNA and lack of receptor agonist binding in spinal cord membranes. Unexpectedly, TRPV3 mRNA expression was upregulated in shRNAtg but downregulated in knockout mice. Capsaicin-induced [Ca(2+)](i) changes in small diameter DRG neurons were significantly diminished in TRPV1 shRNAtg mice, and administration of capsaicin hardly induced hypothermia or nocifensive behaviour in vivo. Likewise, sensitivity towards noxious heat was reduced. Interestingly, spinal nerve injured TRPV1 knockout but not shRNAtg animals developed mechanical allodynia and hypersensitivity. The present study provides further evidence for the relevance of TRPV1 in neuropathic pain and characterizes RNA interference as valuable technique for drug target validation in pain research.

Inducible Transgenic Rat Model for Diabetes Mellitus Based on shRNA-Mediated Gene Knockdown

The rat is an important animal model in biomedical research, but gene targeting technology is not established for this species. Therefore, we aimed to produce transgenic knockdown rats using shRNA technology and pronuclear microinjection. To this purpose, we employed a tetracycline-inducible shRNA expression system targeting the insulin receptor (IR). Doxycycline (DOX) treatment of the resulting transgenic rats led to a dose-dependent and reversible increase in blood glucose caused by ubiquitous inhibition of IR expression and signalling. We could neither detect an interferon response nor disturbances in microRNA processing after DOX treatment excluding toxic effects of shRNA expression. Low dose DOX treatment induced a chronic state of diabetes mellitus. In conclusion, we have developed a technology which allows the specific, inducible, and reversible suppression of any gene of interest in the rat. Our first transgenic rat line generated with this method represents an inducible model for diabetes mellitus.

Architecture and utilization of highly expressed genomic sites

Publisher Summary A major reoccurring problem in molecular biology, bioengineering, and gene therapy is the variable expression of transgenes. One solution to this problem is sought in the use of nonviral episomal (extrachromosomal) vectors that replicate and segregate in synchrony with the host cell. Although progress has recently been reported, the development of this promising system is still in its infancy. If a stable long-term expression is desired, then integrating systems remain standard. According to conventional transfection protocols, integration occurs into random chromosomal loci and at variable copy numbers, which together cause unpredictable gene expression characteristics because of position-dependent inactivation and repeat-induced silencing processes. To circumvent problems of this kind, methodologies have been developed whereby the gene of interest (GOI) can be inserted into a pre-characterized chromosomal site at the location of a unique genomic tag. Retroviral vectors are considered particularly useful vehicles for a GOI because the retroviral integration machinery is well suited to introduce a single intact copy into a subclass of apparently favorable genomic sites. The chapter discusses the architecture of loci enabling stable and high-level expression, the methods for their characterization, and the principles which can be applied to guide a transgene into a characterized site with known properties.

Efficient FACS selection procedure for cells undergoing Flp-mediated site-specific conversions

Michaela Iber is in the National Research Center for Biotechnology, Department of Gene Regulation and Differentiation, Mascheroder Weg 1, D-38124 Braunschweig, GermanyDirk Schubeler is in the National Research Center for Biotechnology, Department of Gene Regulation and Differentiation, Mascheroder Weg 1, D-38124 Braunschweig, GermanyJost Seibler is in the National Research Center for Biotechnology, Department of Gene Regulation and Differentiation, Mascheroder Weg 1, D-38124 Braunschweig, GermanyMaria Hoxter is in the National Research Center for Biotechnology, Department of Gene Regulation and Differentiation, Mascheroder Weg 1, D-38124 Braunschweig, GermanyJurgen Bode is in the National Research Center for Biotechnology, Department of Gene Regulation and Differentiation, Mascheroder Weg 1, D-38124 Braunschweig, Germany