Vector systems

Modified mammalian cell systems for the study of the role of transporters and/or metabolism in oral absorption consists of two main components, the cell line and the 

The key function of the vector is to introduce the cDNA under the control of a strong promoter and, if a stable cell line is to be developed, also to introduce resistance to a compound that is otherwise toxic to the host cell. This facilitates selection (only vector-bearing cells grown in special media) of a minority of cells that have incorporated the vector. There are many adequate expression vectors available from a number of commercial suppliers. Several classes of vector systems appear appropriate for transporter expression as integrating, episomal, retroviral, Vaccinia virus-, and Adenovirus-based systems have all been used successfully in studies referenced in this chapter. 

Alternatively, the transporter function can be analyzed by accumulation or efflux assay. Accumulation assay is generally used to determine substrate accumulation rate into the cells mediated by uptake transporters, whereas efflux assay is generally used to determine substrate efflux rate of the cells mediated by efflux transporters. For efflux assay, cells are preincubated with the substrate. 

For efflux transporters, however, the functional interaction between the drug and the transporter occurs in the intracellular space. Some substrates, particularly the negatively charged MRP2 substrates, have very low permeability and in the absence of an uptake transporter may not give detectable transport. In these cases, double-transfected cell lines have been developed so that compounds with low permeability 

Apical side = Donor Apical side = Receiver 

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J. Vlak and R. Keus, "Baculovims Expression Vector System for Production of Viral Vacciues," iu A. Mizrahi, ed.. Viral Vaccines, Wiley-Liss, New York, 1990. 

LDH-x is one of the best characterized antigens and its amino acid sequence is known. A synthetic peptide based on a portion of the molecule has been shown to reduce fertUity in laboratory animals. The nucleotide sequence coding for human LDH-x has been defined and engineered into an expression vector system (121). 

Another important issue is the size-limitation of AAV vectors the maximum size of rAAV genomes is 4.7 kb. To overcome this size constraint, dual vector systems with split-genomes have been developed [1] dual vectors are based on episomal circular multimers formed by the AAV vector genomes. However, the efficacy of the dual vector system has been questioned. 

To circumvent this problem, vectors that are based on lentiviruses have been developed. In contrast to prototypic retroviruses, lentiviruses do not require cell division for integration. Gene-therapy vectors have been developed from a broad spectrum of lentiviruses including human immunodeficiency vims (HIV), simian and feline immunodeficiency vims as well as visna/maedi vims. The most widely used lentiviral vector system is based on HIV-1. These vectors can efficiently transduce a broad spectrum of dividing and nondividing cells including neurons, hepatocytes, muscle cells, and hematopoietic stem cells [1,2]. 

In conclusion, several vector systems are available in the molecular therapy toolbox, each suited for specific applications. 

Nuclei resonating at different chemical shifts will also experience similar refocusing effects. This is illustrated by the accompanying diagram of a two-vector system (acetone and water), the nuclei of which have different chemical shifts but are refocused together by the spin-echo pulse (M, = magnetization vector of acetone methyl protons, M(v = magnetization vector of water protons). 

Philipps, B., Forstner, M. and Mayr, L.M. (2005) A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells. Biotechnology Progress, 21 (3), 708-711. 

Miller, A.D., The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy, Current Medicinal Chemistry, 2003, 10, 1195-1211. 

Boeckle S, Wagner E (2006) Optimizing targeted gene delivery chemical modification of viral vectors and synthesis of artificial virus vector systems. AAPS J 8 E731-E742... 

EPL extends the applicability of native chemical ligation to recombinantly produced proteins using the mutant mini intein vector system. Proteins being expressed using this method will... 

The design of chimeric viruses to create a functional vector system combining components of different viruses and thus expanding the virus host range was one of the early approaches employed by Yusibov et al. [32]. Antigenic determinants from... 

Mergulhao, F.J.M., Monteiro, G.A., Cabral, J.M.S., and Taipa, M.A. 2004. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. Journal of Microbiology and Biotechnology. 14, 1-14. 

A number of factors render vaccinia virus a particularly attractive vector system. These include ... 

Table 14.2 Vector systems used to deliver genes into mammalian cells3...

Viral-based vector systems Non-viral-based vector systems... 

A list of the various vectors capable of introducing genes into recipient cells has been provided in Table 14.2. These vectors are conveniently categorized as being viral-based or non-viral-based systems. The main vector systems developed, thus far, are discussed in somewhat more detail below. 

A requirement for improved, more target-specific vector systems. 

Holmgren, J., N. Lycke, C. Czerkinsky, Cholera-Toxin and Cholera-B Subunit as Oral Mucosal Adjuvant and Antigen Vector Systems, Vaccine. 11, 1179, 1993. 

Expression of a recombinant protein using an inducible vector system would permit expression at endogenous levels to simulate physiologic levels of expression of a protein of interest. Tandem affinity purification strategies have recently been employed and facilitate the analyses of highly interactive proteins when the bait protein is expressed at endogenous levels. Immunoaffinity or immunoprecipitation followed by LC-MS/MS does not readily permit determination of the stoichiometry of interacting partners. Additionally, when compared to yeast hybrid experiments, it is difficult to determine whether interactions are binary when identified in complexes by MS/MS. 

Formation of a complex between DNA and polycationic compounds appears to be the initial and quite possibly a critical parameter for nonviral gene delivery. Several synthetic vector systems, which are generally cationic in nature, including poly(lysines), cationic liposomes or various types of block copolymers and recently dendrimers, have been shown to self-assemble with plasmid DNA [13-15] [16]. Specific physicochemical properties manifested by these DNA complexes depend on the type of cationic agent used however, interesting patterns for such interactions are beginning to evolve [17, 18]. Under certain conditions, the interaction of DNA with polyvalent cations results in... 

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