scholarly journals Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

2019 ◽  
Vol 30 (22) ◽  
pp. 2761-2770
Author(s):  
Xiao-Yin Wang ◽  
Xi Zhang ◽  
Tian-Yun Wang ◽  
Yan-Long Jia ◽  
Dan-Hua Xu ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Transgene Expression ◽  
Matrix Attachment Regions ◽  
Expression Levels ◽  
Episomal Vector ◽  
Maintenance Mechanism ◽  
Molecular Mode ◽  
Episomal Maintenance

Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.

scholarly journals Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny

1999 ◽  
Vol 18 (3) ◽  
pp. 233-242 ◽  
Author(s):  
Philippe Vain ◽  
Barbara Worland ◽  
Ajay Kohli ◽  
John W. Snape ◽  
Paul Christou ◽  
...  
Keyword(s):  
Transgenic Rice ◽  
Transgene Expression ◽  
Matrix Attachment Regions ◽  
Expression Levels ◽  
Rice Plants ◽  
Transgenic Rice Plants

scholarly journals RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wen Wang ◽  
Yan-long Jia ◽  
Yi-chun Li ◽  
Chang-qin Jing ◽  
Xiao Guo ◽  
...  
Keyword(s):  
Cho Cells ◽  
Transgene Expression ◽  
Recombinant Protein Expression ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Vector System ◽  
Gene Copy ◽  
Elongation Factor 1Α ◽  
Cmv Promoter ◽  
Expression Levels

Abstract In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Matrix attachment regions and regulated transcription increase and stabilize transgene expression

2005 ◽  
Vol 3 (5) ◽  
pp. 535-543 ◽  
Author(s):  
Rita Abranches ◽  
Randall W. Shultz ◽  
William F. Thompson ◽  
George C. Allen
Keyword(s):  
Transgene Expression ◽  
Matrix Attachment Regions

scholarly journals Multiple Effects of Genetic Background on Variegated Transgene Expression in Mice

Genetics ◽  
2002 ◽  
Vol 160 (3) ◽  
pp. 1107-1112
Author(s):  
Margaret L Opsahl ◽  
Margaret McClenaghan ◽  
Anthea Springbett ◽  
Sarah Reid ◽  
Richard Lathe ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Milk Protein ◽  
Position Effect ◽  
Position Effect Variegation ◽  
Parental Population ◽  
Expression Levels ◽  
Variable Expression ◽  
Specific Effects ◽  
Milk Protein Genes ◽  
Β Lactoglobulin

Abstract BLG/7 transgenic mice express an ovine β-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA × C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

scholarly journals Effect of nuclear matrix attachment regions on transgene expression in tobacco plants.

2001 ◽  
Vol 48 (3) ◽  
pp. 637-646 ◽  
Author(s):  
W Nowak ◽  
M Gawłowska ◽  
A Jarmołowski ◽  
J Augustyniak
Keyword(s):  
Transgenic Plants ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Binary Vector ◽  
Special Property ◽  
Matrix Attachment Regions ◽  
Plant Populations ◽  
Tobacco Plants ◽  
Gus Reporter ◽  
Loop Domains

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.

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