Episomal vectors for gene expression in mammalian cells

Craenenbroeck, Kathleen Van; Vanhoenacker, Peter; Haegeman, Guy

doi:10.1046/j.1432-1327.2000.01645.x

Cited by 129 publications

(104 citation statements)

References 188 publications

(256 reference statements)

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“…It shows that moss is capable of faithful episomal replication of foreign DNA without rearrangements. This is unlike episomal vectors in mammalian cells, where rearrangements and insertions of host DNA are frequent (19), and instead resembles yeast shuttle vectors. It does not rule out that replication in moss involves complex intermediates, such as episomal concatemers (15), since the latter could pop out circles by recombination (Fig.…”

Section: Discussionmentioning

confidence: 99%

Rescue and characterization of episomally replicating DNA from the moss Physcomitrella

Murén

Nilsson

Ulfstedt

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The moss Physcomitrella is unique among plants in that it permits efficient gene targeting by homologous recombination. Furthermore, transformed DNA can replicate episomally in Physcomitrella. Here we show that episomally replicating DNA can be rescued back into Escherichia coli, and we use such rescue to study the fate of the transformed DNA. Significantly, plasmids rescued from moss transformed with circular DNA are identical to the original plasmid, whereas plasmids rescued from moss transformed with linearized DNA frequently have deletions created by direct repeat recombination. These events are highly predictable in that they target the longest direct repeat on the plasmid if this repeat is at least 12 bp. Episomal transformants obtained with linearized DNA show a more than 1,000-fold amplification of the DNA whereas transformants obtained with circular DNA have much lower copy numbers. Most episomal transformants quickly lose the plasmid in the absence of selection, but a semistable type of transformant that loses the plasmid at a much lower frequency was also observed. The consistent rescue of the original plasmid, or of predictable derivatives thereof, suggests that molecular genetics methods which rely on shuttle plasmids are feasible in Physcomitrella.episomal replication ͉ plasmid rescue ͉ recombination ͉ shuttle plasmid T he discovery of gene targeting by homologous recombination (1) and the development of yeast shuttle plasmids (2-4) were key advances in eukaryotic molecular genetics. Gene targeting made reverse genetics possible, and shuttle plasmids made it easy to clone both yeast genes and genes from other eukaryotes by complementation of yeast mutants (5). Furthermore, the effects of overexpressing a gene could be studied by using plasmids that replicate in high copy number; by screening genomic libraries made in such plasmids, dosage suppressors of yeast mutants could be cloned, one of the most powerful methods available in molecular genetics (6).The finding that gene targeting is feasible in the moss Physcomitrella (7) transformed this small plant into an important model organism (8-11). Interestingly, early studies also showed that foreign DNA can replicate episomally in Physcomitrella (12-15). Three types of transformants were described by Schaefer (14). Class I have the DNA integrated into the moss genome and further studies of these transformants led to the discovery of gene targeting in moss (7). Class II, which comprise most transformants, quickly lose the marker under nonselective growth. Class III transformants also lose the marker in the absence of selection, but the rate of loss is lower (14). Further studies by Ashton et al. (15) showed that foreign DNA can replicate episomally in moss for at least 10 years if selection is maintained. Southern blots and PCR experiments suggested that the episomal DNA forms concatemers with 3-40 copies of the plasmid (14, 15). A notable finding in both studies was that moss DNA does not seem to be required for replication, unlike the case in yeast....

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Section: Discussionmentioning

confidence: 99%

Rescue and characterization of episomally replicating DNA from the moss Physcomitrella

Murén

Nilsson

Ulfstedt

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Cloning and Expression of HtrA1 Recombinant Proteins-Recombinant murine HtrA proteins were produced using a mammalian episomal expression vector (pCEP-His) and 293-EBNA cells (29). pCEP-His has been modified from the pCEP-pu/AC7 vector (30) to incorporate an N-terminal His 6 tag following a signal peptide of the BM40 protein (31).…”

Section: Methodsmentioning

confidence: 99%

HtrA1 Inhibits Mineral Deposition by Osteoblasts

Hadfield

Rock

Inkson

et al. 2008

Journal of Biological Chemistry

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HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.Mammalian HtrA1 is a member of the family of HtrA (high temperature requirement) proteins that were originally identified in bacteria (1). These proteins are characterized by the presence of a trypsin-like serine protease domain and either one or two PDZ domains. However, in contrast to bacterial HtrA, mammalian HtrA1 is secreted, and it also contains an IGFBP 4 /mac25-like domain and a Kazal-type inhibitor domain at the N terminus (2, 3). Evidence is now accumulating to suggest that HtrA1 is involved in the development and progression of several pathologies. HtrA1 has been found to exhibit properties of a tumor suppressor protein, as its expression is downregulated in many cancer cell lines (2, 4), low levels of HtrA1 are detected in cancerous tissue compared with normal tissues (4, 5), and overexpression of this protein inhibits tumor cell growth in vivo and in vitro (4, 5). HtrA1 expression is also markedly up-regulated in several diseases, including rheumatoid arthritis and osteoarthritis (3, 6 -8), Alzheimer disease (9), and Duchenne muscular dystrophy (10). In addition, a single-nucleotide polymorphism in the gene encoding HtrA1 has been shown to increase susceptibility to age-related macular degeneration (...

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“…The generation of artificial chromosomes with an optimal size with respect to stability as well as transfection efficiencies, can be promising for the future of nonviral gene therapy as it may form the perfect alternative for viral episomal gene vectors (see next paragraph). This is even more interesting in the context of possible immunogenic or oncogenic properties of viral vectors [171]. Additionally, the transgenes bear strong constitutive promoters, which result in nonphysiological overexpression.…”

Section: Nonviral Chromatin Tethering Factorsmentioning

confidence: 99%