Minicircle performance depending on S/MAR-nuclear matrix interactions.

The ideal vector for cell and tissue modification does not depend on integration but rather behaves as an independent functional unit that replicates as an episome. Based on a scaffold/matrix attachment region (S/MAR), we have introduced, in 2006, an approximately 4-kb replicating nonviral minicircle able to exploit the cellular replication machinery in a way reminiscent of ARS vectors. Consisting of only one active transcription unit and the S/MAR, it resists silencing as it is free of prokaryotic vector parts and drug selection markers. The rate of final establishment in the nuclear architecture is moderate but comparable to Epstein-Barr virus-based episomes (<5%). Here, we demonstrate that this parameter can be improved if the host cell chromatin is opened by histone hyperacetylation prior to transfection. It remains unaffected, however, by cell cycle position. Still, this class of episomes revealed intrinsic instability and integration after 5 months of continuous culture. In vivo evolution enabled the effective reduction of S/MAR size from 2 kb to 733 bp (resulting in a minicircle of approximately 3 kb) with largely improved stability and cloning capacity. Investigation of individual clones served to prove persistent and homogenous expression, which is ascribed to stable association with nuclear attachment sites. Optimum expression levels were shown to depend on the authentic usage of a polyadenylation site 3' from the S/MAR as anticipated by the stress-induced duplex destabilization algorithm, which finds increasing use to predict the functional parameters of these systems.

Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties.

Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.

PARP-1 expression in the mouse is controlled by an autoregulatory loop: PARP-1 binding to an upstream S/MAR element and to a novel recognition motif in its promoter suppresses transcription.

This work identifies central components of a feedback mechanism for the expression of mouse poly(ADP-ribose) polymerase-1 (PARP-1). Using the stress-induced duplex destabilization algorithm, multiple base-unpairing regions (BURs) could be localized in the 5' region of the mouse PARP-1 gene (muPARP-1). Some of these could be identified as scaffold/matrix-attachment regions (S/MARs), suggesting an S/MAR-mediated transcriptional regulation. PARP-1 binding to the most proximal element, S/MAR 1, and to three consensus motifs, AGGCC, in its own promoter (basepairs -956 to +100), could be traced by electrophoretic mobility-shift assay. The AGGCC-complementary GGCCT motif was detected by cis-diammine-dichloro platinum cross-linking and functionally characterized by the effects of site-directed mutagenesis on its performance in wild type (PARP-1(+/+)) and PARP-1 knockout cells (PARP-1(-/-)). Mutation of the central AGGCC tract at basepairs -554 to -550 prevented PARP-1/promoter interactions, whereby muPARP-1 expression became up-regulated. Transfection of a series of reporter gene constructs with or without S/MAR 1 (basepairs -1523 to -1007) and the more distant S/MAR 2 (basepairs -8373 to -6880), into PARP-1(+/+) as well as PARP-1(-/-) cells, revealed an additional, major level of muPARP-1 promoter down-regulation, triggered by PARP-1 binding to S/MAR 1. We conclude that S/MAR 1 represents an upstream control element that acts in conjunction with the muPARP-1 promoter. These interactions are part of a negative autoregulatory loop.

NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.

The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology.

The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like PhiC31, which recombine attP and attB target sites in a one-way reaction - at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and PhiC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is 'clean' in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.

Nucleolar localization and mobility analysis of the NF-kappaB repressing factor NRF.

NF-kappaB plays a central role in mediating pathogen and cytokine-stimulated gene transcription. NF-kappaB repressing factor (NRF) has been shown to interact with specific negative regulatory DNA elements (NRE) to mediate transcriptional repression by inhibition of the NF-kappaB activity at certain promoters. mRNA ablation experiments demonstrated that the trans-acting NRF protein is involved in constitutive but not post-stimulated silencing of IFN-beta, IL-8 and iNOS genes by binding to cis-acting NRE elements in their promoters. We have examined the subcellular localization and mobility of the NRF protein. Since neither tagging nor overexpression perturbs NRF localization the GFP-tagged protein was used for detailed localization and mobility studies. Owing to an N-terminal nuclear localization sequence, all NRF fragments that contain this signal show a constitutive nuclear accumulation. C-terminal NRF fragments also localize to the nucleus although no canonical NLS motifs were detected. Full-length NRF is highly enriched in nucleoli and only a small fraction of NRF is found in the nucleoplasm and cytoplasm. This relationship was found to be independent of the protein expression rate. FRAP analysis proved to be a sensitive method to determine protein mobility and made it possible to differentiate between the NRF protein fragments. Nucleolar localization correlated inversely with mobility. The data demonstrate that a series of neighboring fragments in a large central domain of the protein contribute to the strong nucleolar affinity. These properties were not altered by viral infection or LPS treatment. Several sequence motifs for RNA binding were predicted by computer-mediated databank searches. We found that NRF binds to double stranded RNA (dsRNA). This property mapped to several NRF fragments which correlate with the nucleolar affinity domain. Since treatment with actinomycin D releases NRF from nucleoli the identified RNA binding motifs might act as nucleolar localization signals.