Properties of plasmid vector

After uptake of the plasmid, the protein is produced endogenously and processed intracellularly into small antigenic peptides by the host proteases. One further feature is essential for a functionally useful vector, and that is a selectable marker. Plasmid uses the same enzymes needed for replication of the yeast chromosomes. Persistent transgene expression has been shown in rodents and larger animals years after a single injection of AAV.New vectors have been engineered to enable increased long-term expression. The most fundamental property of a plasmid, whether we are considering it as a cloning vector or as a natural phenomenon, is therefore the ability to replicate in the host bacterium. Indirect IC delivery, instead, has been used to deliver virus vectors to the left ventricle in mice and rats. This does not affect the function of the enzyme; it is still able to hydrolyse lactose. This is done by synthesizing a short piece of DNA with the required restriction sites, and inserting that into the plasmid in the usual way. Wild-type ColEl is present at about 15 copies per cell, while most of the engineered vectors used today are present in numbers running into many hundreds of copies per cell. The most fundamental property of a plasmid, whether we are considering it as a cloning vector or as a natural phenomenon, is therefore the ability to replicate in the host bacterium. However, since the recombinant plasmid now has two BamHI sites, it would be difficult to clone further BamHI fragments into it.The problem here is that in many cases we do want to insert several fragments into the same plasmid. Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication. Therefore in order to be able to recover the transformed clones, it is necessary to be able to prevent the non-transformed cells (i.e. We will be coming across various applications of shuttle vectors in subsequent chapters.Therefore, the first essential characteristic of a plasmid cloning vector is the origin of replication, usually designated as ori in plasmid maps.The second characteristic that is necessary for a plasmid to be useful as a cloning vector is a cloning site. This is a disadvantage, because you are likely to want to use E. coli as an intermediate host for the initial cloning and for studying the structure and behaviour of the gene that you have cloned. Adenovirus provides reasonable gene transfer efficiency,AAV, with an insert capacity <5 kb, provides potential long-term expression. A basic plasmid used as a cloning vector may contain only one or two such unique restriction sites, which must of course be located in a region of the plasmid that is not essential for replication or any other functions that we need.With such a plasmid you are limited not only in your choice of restriction fragments that can be inserted and in the position of insertion, but also in the number of different fragments that can be inserted. The product of the host gene is unable to hydrolyse lactose by itself, and so the host strain without the plasmid is Lac-, i.e. This is because in most cases ligation of two restriction fragments, generated with the same enzyme, recreates the original restriction site. It will then usually be necessary to isolate or construct new vector plasmids, based on a replication origin that is functional in your chosen species. On the other hand, the basic plasmid vectors that we have been considering so far are limited in their cloning capacity, i.e. There are four regulated expression systems currently available: ecdysone, tamoxifen, tetracycline, and rapamycin.AP22594, oral rapamycin analog with 1/100th immune suppression versus rapamycin.The tetracycline-regulation system has been extensively studied.Immune responses to components of the rtTA system did not occur when AAV4.tet and AAV5.tet gene transfer (intraretinal) were used in nonhuman primates,In the rapamycin regulation system, transgene expression is triggered by nanomolar concentrations of rapamycin or a rapamycin analog, which is dose-dependent and reversible.Gene transfer of peptides with paracrine activities that may benefit the heart is an alternative to cardiac-targeted gene transfer and may be applicable for CHF and other cardiovascular diseases.

You can make and use plasmid vectors for a wide range of organisms without a detailed knowledge of the molecular biology of the host or the vector. It provides strong transgene expression in cardiac myocytes.