Vector design

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In genetic engineering, the term vector design refers to the targeted adaptation of a vector using molecular biological methods for the replication of DNA . The plasmid pBR322 was developed from a naturally occurring plasmid as early as 1977 . As a result, this and other plasmids were reduced in size in the course of the vector design and given new antibiotic resistances and origins of replication . The most frequently modified section often involves the cloning site, an area where many once in a plasmid occurring ( unique ) restriction enzyme recognition sites are, the multiple cloning site ( mcs , multiple cloning site ), even polylinker called. It is used for the targeted integration of DNA molecules, with the aim of duplicating (amplifying) foreign DNA. To generate recombinant proteins , coding or non-coding ribonucleic acids and their in vitro or in vivo use, suitable flanking regulatory sites are inserted. This is done, for example, for transient purposes such as vaccines (e.g. transient viral vectors or DNA vaccines ), transient transfection or in the case of oncolytic viruses, and for permanent purposes such as gene therapy or the generation of genetically modified organisms .

The organism or cell type during production is not necessarily the target organism of the vector ( shuttle vector , e.g. plasmids with eukaryotic expression cassettes , viral vectors for packaging in cell cultures ). Often, therefore, adaptations are made to both types of hosts .

Process and Effects

To increase gene expression , the DNA sections outside of the protein-coding sequence are also changed. The choice of promoter , enhancer and terminator can increase the amount of expression provided that they can be used in the species used for protein expression. There are clear differences between mammals, bacteria and archaea. The vector design also depends on the use as a cloning vector or as an expression vector .

In eukaryotes, nuclear export sequences can increase the RNA concentration in the cytosol and thus the amount of expression by the ribosome there . Furthermore, the recognition of the mRNA on the ribosome can be improved by a Kozak sequence . A polyadenylation signal and the avoidance of AUUUA sequences at the 3 'end can reduce the premature degradation of the mRNA. The modular property of the polylinker can be modified by an insertion or deletion of a recognition sequence for restriction enzymes . Occasionally, modular interchangeable expression cassettes are used which allow an exchange by homologous recombination or by the RMCE cassette exchange method . When expressing several proteins, several vectors, several operons in one vector, IRES , inteins or 2A peptides can be used.

The replication of a plasmid vector can be extended in several ways by inserting one or more origins of replication and possibly necessary centromeres , whereby longer-term expression can be achieved with plasmids, e.g. B. plasmids in mammalian cells.

The modification of the protein-coding sequence is usually carried out in parallel with the vector design. In the course of a protein design , e.g. B. through a codon optimization of the expression cassette, the expression rate can be increased or other properties can be changed.

Vector security

A key requirement for a vector is the safety of its use in living beings, which is characterized by the biological protection level in relation to humans . The classification of the protection level considers the pathogenicity , which is determined, among other things, by the possibility of replication , a permanent change in the genome (the insertion promotes tumor development ), as well as by the infectivity and the symptoms .

In order to avoid an excessive immune reaction , production- related pyrogens are examined in bacterially generated vectors . In the case of plasmids, the antibiotic resistance- mediating resistance genes , which are used for selection during production , must not be transferred to the microorganisms (e.g. skin flora , oral flora and intestinal flora ). Therefore, as an alternative, auxotrophies are used to select the transgenic organisms or the resistance genes are subsequently removed.

In the case of viral vectors , some genes necessary for replication are removed by deletion , so that generation can only take place by complementation in a cell line that has previously received these genes. This is known as replication deficiency. Likewise, by changing the receptor, the tropism can be limited or changed in the course of a pseudotyping . By using cell-type- specific promoters, gene expression can be localized .

Prior to the development of genetic engineering methods, an increase in the security of a virus was achieved through attenuation . These live vaccines include e.g. B. the oral vaccination against poliomyelitis or the vaccinia virus- based smallpox vaccines , MVA and NYVAC.

literature

Individual evidence

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  7. M. Kozak: An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. In: Nucleic Acids Res . (1987) Vol. 15 (20), pp. 8125-48. PMID 3313277 ; PMC 306349 (free full text).
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