Biological Sciences
Expression Vector
An expression vector is a type of DNA molecule used to introduce a specific gene into a target organism for the purpose of expressing the gene and producing its corresponding protein. These vectors typically contain regulatory elements that control the expression of the gene, as well as selectable markers for identifying cells that have successfully taken up the vector.
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8 Key excerpts on "Expression Vector"
eBook - PDF- Moses P. Adoga(Author)
- 2012(Publication Date)
- IntechOpen(Publisher)
Main viral vectors exploited for use in neurobiology are adeno-associated vectors, herpes simplex vectors and lentiviral vectors. Each has both advantages and disadvantages depending basically on the subset of target cells and the specificities of each research or clinical indication. Viral Vectors in Neurobiology: Therapeutic and Research Applications 89 The spectrum of research and therapeutic applications of viral vectors increase to the extent that advances are made in this area. Currently, these applications include functional studies by gene expression in vitro and in vivo ; neuronal tracing, allowing unraveling anatomical neuronal interconnections; neuromodulation, using optogenetic tools and gene therapy. Nevertheless, for each approach there are still a lot of technical hurdles to overcome. However, advancement in this field will most likely lead to new tools being created with the ability to overcome current limitations. 4. Glossary Term Definition Gene expression The process of formation of messen g er RNA (mRNA) of a DNA template which then is translated into the se q uence of aminoacids at the ribosome to make p roteins. Transgene A forei g n g ene introduced into the cell ( for exam p le b y a viral vector ) . Transfection Process of deliverin g a forei g n g ene into the tar g et cell, b y a non-viral vector or naked DNA. Transduction Process of deliverin g a forei g n g ene into the tar g et cell, b y a viral vector. Expression cassette A piece of DNA containin g elements (promoter, codin g part and pol y aden y lation si g nal ) necessar y for ex p ression of a trans g ene. Promoter Re g ion of DNA that facilitates the transcription of a particular g ene. Promoters are located near the g enes the y re g ulate, on the same strand and t y picall y u p stream ( towards the 5’ re g ion of the sense strand ) .
- Ajit S. Narang, Ram I. Mahato, Ajit S. Narang, Ram I. Mahato(Authors)
- 2010(Publication Date)
- CRC Press(Publisher)
This concept has been used to create promoters with combinations of regulatory sequences. Li et al. randomly assembled muscle-specific elements from four different muscle-spe-cific promoters and then screened these novel promoters for activity. 61 One sequence showed 8-times the activity of natural muscle promoters. However, this was done in vitro . It is more challenging to develop a promoter with greater expression in vivo . 92 Targeted Delivery of Small and Macromolecular Drugs 5.2.4 P ULSATILE G ENE E XPRESSION A gene switch is introduced into an expression system to make use of the circadian rhythm by extra-neously administering a compound to control the production of therapeutic proteins by turning on or off the transcription of an administered gene. Gene switches are incorporated into the vector for gene regulation. This feedback control may prevent overexpression and possible deleterious protein production. In this system, the target gene is inactive until the administration of an exogenous com-pound or ligand. 62,63 Progesterone antagonists, tetracycline, ecdysone, and rapamycin are used as inducing agents to turn on the gene regulation of the Expression Vector. 5.3 VIRAL VECTORS Recombinant viruses are unique in being naturally evolved vehicles that efficiently transfer their genes into host cells. Viral vectors are composed of either an RNA or a DNA core, or process dif-ferent genomic structure and host ranges. However, they often have risks such as toxicity, immuno-genicity, and/or potential for viral recombination. The novel developments of viral vectors mainly aim at the reduction of immunogenicity and improved vector production. Several kinds of viruses including retrovirus, Adv, AAV, and herpes simplex virus (HSV) have been manipulated for gene transfer. These viral vectors have their own unique advantages and disadvantages as discussed below. 5.3.1 R ETROVIRAL V ECTORS Retroviruses are enveloped single-stranded RNA viruses.
- Khushboo Chaudhary(Author)
- 2019(Publication Date)
- Delve Publishing(Publisher)
Tools of Genetic Engineering (Cloning Vectors) 5 5.1. CLONING AND Expression VectorS In recent years, techniques for manipulating prokaryotic as well as eukaryotic DNA have witnessed a remarkable development. This has allowed breakage of a DNA molecule at two desired places to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position. The product thus obtained is called recombinant DNA and the techniques often called genetic engineering . Using, this technique we can isolate and clone single copy of a gene or a DNA segment in to an indefinite number of copies, all identical. This became possible because vectors like plasmids and phages reproduce in a host in their usual manner ever after insertion of foreign DNA, so that the insertion DNA will also replicate faithfully with the parent DNA. This technique is called gene cloning. With this technique, genes can be isolated, cloned and characterized, so that the technique has led to significant progress in all areas of molecular biology. A variety of vectors have been developed which not only allow multiplication, but may also be manipulation in such a way that the inserted gene may express in the host. Due to the importance of a variety of these cloning and Expression Vectors in genetic engineering experiments, they are discussed in some detail in this chapter. The techniques used for inserting foreign DNA molecules will be discussed in next chapter. The commercial use of these techniques will be discussed in subsequent chapters. 5.2. CLONING VECTOR FOR RECOMBINANT DNA One of the most important use of recombinant DNA technology in the cloning of (i) random DNA or cDNA segments, often use as probe or (ii) specific genes, Introduction to Biotechnology and Biostatistics 132 which may be either isolated from the genome or synthesized organochemically or in the form c DNA from m RNA.
eBook - PDFBiosimilars and Interchangeable Biologics
Tactical Elements
- Sarfaraz K. Niazi(Author)
- 2016(Publication Date)
- CRC Press(Publisher)
Vectors Eukaryotic Expression Vectors are of two basic types: virion or virion-plasmid hybrids. Virion-type vectors are most commonly used for the delivery of foreign genes, or a replacement for a defective host gene, into mammalian cell hosts. The virion-plasmid hybrid vectors are used to facilitate the overexpression of protein in native form. Additionally, the availability of authentic pure protein has hastened the development of structure-dependent epitope-specific antibodies to native pro-teins, the crystallization and subsequent x-ray/NMR analysis of proteins in their native, correctly folded, posttranslationally modified state, the characterization of gene products for genes whose phenotype may or may not be known, and the iso-lation of proteins whose native form is in such low abundance that they are very difficult to purify from the original natural organism or tissue. The main features of eukaryotic Expression Vectors include various sequence elements that explicitly define the level of expression, the transcriptional start and stop points, postprocess-ing (transcript splicing, polyadenylation, etc.) transport, selectable markers, and in some cases a peptide tag to facilitate isolation and purification of the gene product. The earliest Expression Vectors, pSV2, is a composite of sequence elements from the papova virus, Simian Virus 40 and the prokaryotic cloning vector, pBR322. SV40 sequence provided transcriptional enhancers, promoter (early region), splic-ing signal (small t-antigen gene), and the polyadenylation signal element, and pBR322 provided the origin of replication (ori) element. The presence of a select-able marker, pBR322 AmpR gene, completes this model eukaryotic Expression Vector. Practically all modern eukaryotic Expression Vectors possess one or more of these elements. Other viruses used include cytomegalovirus (CMV), Murine sarcoma virus (MSV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), and Semliki Forest virus (SFV).
eBook - ePub- S.C. Makrides(Author)
- 2003(Publication Date)
- Elsevier Science(Publisher)
12 ]. This approach exploits the cell-targeting specificity of viruses without the disadvantages of virus-based vectors.4 Genetic elements of mammalian Expression Vectors
Vectors for protein production in mammalian cells comprise a variety of genetic elements with distinct functionalities (Fig. 1 ): (1) a constitutive or inducible promoter that is capable of robust transcriptional activity; (2) a transcription terminator that stabilizes the transcript and prevents transcription interference; (3) optimized mRNA processing and translational signals that include the Kozak sequence, translation termination codon, mRNA cleavage and polyadenylation signals, as well as mRNA splicing signals for higher levels of expression; (4) prokaryotic origin of replication and selection marker for vector propagation in bacteria; and (5) selection markers for the preparation of stable cell lines and for gene amplification. The inclusion of the SV40 origin of replication facilitates transient gene expression in COS cells. Other genetic elements for specific applications include sequences for gene or protein targeting, signal peptides for protein secretion, fusion moieties and protease cleavage sites (see Section 6 ), and ribosome- or protease-recognition sites that facilitate the expression of multiple genes from polycistronic (Fig. 1 C) or monocistronic (Fig. 1 D) constructs, respectively (see Section 8 ). An extensive list of mammalian Expression Vectors has been published [1 ].Fig. 1 Configuration of model genetic elements in mammalian Expression Vectors. The combination of different elements (not drawn to scale) may vary in order to meet specific objectives. SV40 ori facilitates transient gene expression in COS cells. Promoters (P) facilitate constitutive (A) or inducible (B) expression. The optimal translational initiation sequence (Kozak) and termination tetranucleotide are shown. The ColE1 origin and the Ampr gene allow plasmid replication and selection, respectively, in bacteria. The Neor gene facilitates selection, and the dhfr gene allows both selection and gene amplification in cells. Multiple gene expression utilizes polycistronic constructs (C) where IRES elements enable ORFs to be translated from a single transcript (see Section 8 ). Alternatively, a monocistronic construct (D) contains in-frame cDNAs joined by linkers encoding recognition sites (Arg-X-Arg/Lys-Arg) for the endoprotease furin, thus facilitating the post-synthetic cleavage of different proteins (see Section 8 ). Abbreviations : Ampr , ampicillin-resistance gene (β-lactamase); ColE1, prokaryotic origin of replication; dhfr, dihydrofolate reductase (methotrexate resistance); F, furin-recognition sequence; FUS, fusion moiety; hCMV-IE, human cytomegalovirus immediate early enhancer/promoter; IRES, internal ribosome entry site; L, leader (targeting sequence); MCS, multiple cloning site; Neor , neomycin-resistance gene (aminoglycoside phosphotransferase, aph
eBook - PDF- (Author)
- 2014(Publication Date)
- Academic Press(Publisher)
/. Design of an Expression Vector The Expression Vectors used in this work are derivatives of PBR322 1 containing a 247-bp DNA fragment carrying the leftward operators and leftward promoter (P L ) of phage ë. 2 This strong promoter is tightly regu-lated by a repressor protein, product of the phage gene el. Mutants are available that synthesize a thermolabile repressor. 3 This property allows the control of the activity of the P L promoter by a simple temperature shift. The P L expression plasmids 2 are propagated at 28° in E. coli strains which synthesize a temperature-sensitive repressor. While repression of the P L promoter is virtually complete at 28°, full activity is obtained by raising the temperature to 42°. The ability to regulate expression is an important feature of an expression system since continuous high-level expression of an adventitious protein may be lethal to the cell. 4 The cl repressor product can be supplied to the system in a variety of ways. Strains of E. coli are available which synthesize a thermolabile repressor from a defective prophage, e.g., strain Ê12ÄÇ1Ä/ö. 5 Alternatively, the mutant cl gene can be present on a compatible plasmid such as pRK248cIts, 5 pcI857, 6 or on an F' episome. 7 The dual plasmid system allows the use of essentially any E. coli strain as a host for P L Expression Vectors. This versatility is of special interest, as it may be that proper choice of the host results in elevated levels of accumulation of a given protein. While the presence of a strong regulatable promoter on an ex-pression plasmid generally ensures efficient transcription of cloned DNA sequences, efficient translation into protein depends on the presence on the cloned sequence of a ribosome-binding site that is recognized by the E. coli host. As outlined in the Introduction, eukaryotic genes are unlikely 1 F. Bolivar, R. L. Rodriguez, P. Y. Greene, M. C. Betlach, H. L. Heynecker, H. W. Boyer, Y. H. Crosa, and S. Falkow, Gene 2, 95 (1977).
- Hansjörg Hauser, Roland Wagner, Hansjörg Hauser, Roland Wagner(Authors)
- 2011(Publication Date)
- De Gruyter(Publisher)
With this goal in mind, it was impossible to go into the details of most systems. However, we hope that this introduction to the field will help the interested reader to select a system most suitable for his or her purpose. For a detailed discussion of the properties of the individual systems we must refer to more specialized review articles. Detailed reviews of several viral and other expression systems have been published. Several systems, e. g. the adeno-associated virus, were not discussed because their suitability for biotechnology has not yet been shown. The most important achievements in the field of foreign gene expression in animal cells were connected with extensive studies on the molecular biology of viruses. Thus, it seems not unlikely that other viral systems will be explored for the application to foreign gene expression. It also seems possible that improved cell-free expression systems will soon compete with the viral systems. 1.3 Vectors for Gene Transfer and Expression in Animal Cells 79 References Adams, A. (1987) Replication of latent Epstein-Barr virus in Raji cells. J Virol 61,1743-1746. Alkatib G. and Briedis, D.J. (1988) High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system. J. Virol. 62, 2718-2727. Armentano, D., Sookdeo, C.C., Hehir, K.M., Gregory, R.J., St George, J.Α., Prinz, G.A., Wadsworth, S.C. and Smith, A. E. (1995) Characterisation of an adenovirus gene transfer vector containing an E4 deletion. Human Gene Therapy 6, 1343-1353. Artelt, R, Grannemann, R., Stocking, C., Friel, J., Bartsch, J. and Häuser, Η. (1991) The prokaryotic neomy cin-resistance-encoding gene acts as a transcriptional silencer in euka-ryotic cells. Gene 99, 249-254. Aruffo, A. and Seed, B. (1987) Molecular cloning of a CD28 DNA by a high-efficiency Cos cell expression system. Proc. Natl. Acad. Sei. USA 84, 8573-8577. Beames, B., Braunagel, S., Summers, M.D.
eBook - PDF- Xu-bo Yuan(Author)
- 2011(Publication Date)
- IntechOpen(Publisher)
1 Non-Viral Gene Therapy Vectors Carrying Genomic Constructs George Kotzamanis, Hassan Abdulrazzak, Athanassios Kotsinas and Vassilis G. Gorgoulis University of Athens, Medical School Greece 1. Introduction Gene therapy is the use of genes or DNA for the treatment of diseases. For the treatment of inherited disorders, DNA carrying a functional gene is introduced into the cells of a patient to reverse the defect of the corresponding malfunctioning endogenous gene. Previous genetic characterization of the disease and cloning of the gene that causes it are necessary. In most cases, the cDNA of the therapeutic gene is cloned into a bacterial plasmid under the control of a strong heterologous promoter (often of viral origin). However, such constructs, called mini-genes, lack introns, promoters, enhancers, and long-range controlling elements that precisely control the temporal and spatial expression of the endogenous gene. For gene therapy of some diseases it is important to achieve expression of the therapeutic gene at specific levels. Expression at lower levels than normal might not be sufficient to correct the defect and at higher levels could result in undesirable effects. In other cases, tissue-specific expression may be very important. The elements responsible for controlled and tissue-specific expression of a gene usually lie within the introns and the sequences before and after the gene. Therefore, the use of genomic constructs which contain the introns and flanking DNA of the therapeutic gene is expected to be more effective than that of mini-gene constructs in gene therapy for certain genetic diseases where precise levels of the gene product are required (reviewed by (Pérez-Luz & Díaz-Nido, 2010)). Bacterial Artificial Chromosomes (BACs), originating from the human genome project, contain genomic loci of approximately 180 kb on average and cover the entire human genome (Osoegawa et al., 2001).
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