Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Exactly how these epigenetic modifications are directed to the particular gene and the local chromatin has remained enigmatic. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications.

In human cells RNA can specifically direct epigenetic modifications to targeted loci (the promoter regions) and modulate silencing. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by the antisense strand of small-interfering RNAs (siRNAs). These recent observations represent a paradigm shift in which a hidden layer of complexity is involved in gene regualtion and is operative via the action RNA essentially epigenetically regulating DNA.

from Kevin V. Morris (2008). RNA Mediated Transcriptional Gene Silencing. In: Morris, K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Further reading:

1. Epigenetics
2. RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity
3. Molecular Biology Books

Labels: epigenetics, expression, gene regulation, regulation, RNA, RNAi, siRNA

Designed molecules that recognize specific sequences within chromosomal DNA could provide useful probes for natural cellular processes, tools for laboratory experimentation, and lead compounds for therapeutic development. It was discovered that duplex DNA could be recognized by conjugates consisting of DNA oligonucleotides and cationic proteins or peptides. Similarly efficient recognition by neutral peptide nucleic acids (PNAs) was observed. It was found that duplex RNAs could also mediate efficient recognition of duplex DNA. RNAs can target transcription start sites and either inhibit or activate gene expression. This indicates that promoter-targeted RNAs can be powerful tools for regulating gene expression.

from Corey, DR (2008) RNA-Mediated Recognition of Chromosomal DNA. In: Morris , K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Labels: expression, gene regulation, PNA, promoter, regulation, RNA, therapeutics

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. While chromatin remodeling and DNA methylation have been studied for several years now far less is know about how these epigenetic marks are directed to each particular gene. Recently, however the role of RNA in epigenetic gene regulation has begun to become apparent.

from Kevin V. Morris in "Chapter 2 Epigenetic Regulation of Gene Expression" from RNA and the Regulation of Gene Expression

Further reading:

1. RNA and the Regulation of Gene Expression
2. Epigenetics

Labels: epigenetics, expression, regulation, RNA

Many attempts to construct different expression vector systems for mammalian cells have been made in recent years. These vector systems can be categorized in terms of vector administration, mechanisms of vector replication and mechanisms to achieve nuclear persistence of the vectors.

Episomal vectors, either based on viral plasmid replicons or on chromosomal elements, are invaluable tools for basic and applied science. The biochemistry and cell cycle dependent regulation of mammalian DNA replication has been extensively studied using SV40-based vectors and more recently, EBV-based vectors have been used to isolate putative mammalian origins of replication. The non-viral vectors whose functioning depends on the insertion of a chromosomal S/MAR sequence represent a minimal system to study the epigenetic regulation of mammalian DNA replication and the relevance of global functional nuclear architecture. The construction of mammalian artificial chromosomes has had a considerable impact on our understanding of the functional elements of the eukaryotic chromosome, telomeres, replication origins and centromeres. Similarly, the analysis of the biochemistry and regulation of transcription was greatly facilitated by the use of episomal vectors. These constructs will find even further applications for the analysis of basic biological phenomena, such as protein-protein interactions, signal transduction pathways, cell movements and many more.

Long term expression of transgenes in the absence of selection has been reported for EBV-based vectors, S/MAR-based vectors and mammalian artificial chromosomes, making these vectors attractive systems for the production of pharmaceutical relevant proteins in mammalian cells. Mammalian artificial chromosomes and S/MAR-based vectors have been successfully used for the genetic modification of mammalian organisms, such as mice and pigs, and have proven to be significantly more efficient than currently used integrating vectors.

Episomal vectors and especially vectors based on chromosomal elements may represent alternatives to the currently used viral vectors for genetic therapy. The major risks associated with virus-based vectors involve problems associated with insertional mutagenesis, imunogenicity and cytotoxicity. These problems were highlighted by the first therapy related fatality, in which death was caused by an inflammatory reaction that was attributed to the use of adenoviral vectors. Insertional mutagenesis events related to the use of integrating vectors have been reported for retroviral as well as AAV vectors. Since episomal vectors based exclusively on chromosomal elements do not need any virus-encoded protein for their function and furthermore do not integrate into the host cell genome, they should avoid the major safety risks that have been associated with virus-based vectors. Their use has been demonstrated in vitro and some have been propagated in animal systems. Their present main limitation is their low transfection efficiency compared to systems based on replication deficient viruses. However, recent success in the construction of non-viral episomal vectors and steady improvement of DNA transfection techniques makes it likely that these limitations can be overcome in the near future, so that optimized episomal vectors will be available for clinical trials.

fromBaiker et al in Chapter 3. Plasmids: Current Research and Future Trends

Further reading: Plasmids: Current Research and Future Trends

Labels: expression, plasmid, plasmids, vector