...

Expression Vectors in Eukaryotic Cells

by taratuta

on
Category: Documents
71

views

Report

Comments

Transcript

Expression Vectors in Eukaryotic Cells
Page 784
Figure 18.20 Construction of a bacterial expression vector. A cDNA coding region of a protein of interest is inserted downstream of bacterial regulatory sequences (promoter, P) for the lacZ gene, the coding sequence for the mRNA Shine–Dalgarno sequence, the AUG codon, and a few codons for the N­terminal amino acids of the lacZ gene protein. The mRNA produced from this expression vector will therefore direct synthesis of a foreign protein in the bacterium with a few of its N­terminal amino acids of bacterial protein origin (a fusion protein).
18.12— Expression Vectors in Eukaryotic Cells
Mammalian genetic diseases result from missing or defective intracellular proteins. To utilize recombinant techniques to treat these diseases, vectors have to be constructed that can be incorporated into mammalian cells. In addition, these vectors have to be selective for the tissue or cells containing the aberrant protein. Numerous vectors permit the expression of foreign DNA genes in mammalian cells grown in tissue culture. These vectors have been used extensively for elucidation of the posttranslational processing and synthesis of proteins in cultured eukaryotic cells. Unfortunately, the goal to selectively express genes in specific tissues or at specific developmental stages within an animal has met with very limited success.
Several types of expression vectors have been developed that allow the replication, transcription, and translation of foreign genes in eukaryotic cells grown in vitro, including both RNA and DNA viral vectors that contain a foreign DNA insert. These viral vectors are able to infect and then replicate in a host cell. Experimentally constructed vectors that contain essential DNA elements, usually derived from a viral genome, permit expression of foreign gene inserts. Shuttle vectors contain both bacterial and eukaryotic replication signals, thus permitting replication of the vector in both bacteria and mammalian cells. A shuttle vector allows a gene to be cloned and purified in large quantities from a bacterial system and then the same recombinant vector can be expressed in a mammalian cell. Some expression vectors become integrated into the host cell genome while others remain as extrachromosomal entities (episomes) with stable expression of their recombinant gene in the daughter cells. Other expression vectors remain as episomal DNA, permitting only transient expression of their foreign gene prior to cell death.
Foreign DNA, such as viral expression vectors, may be introduced into the cultured eukaryotic cells by transfection, a process that is analogous to transformation of DNA into bacterial cells. The most commonly employed
Page 785
transfection methods involve the formation of a complex of DNA with calcium phosphate or diethylaminoethyl (DEAE)­dextran, which is then taken up by the cell by endocytosis. The DNA is subsequently transferred from the cytoplasm to the nucleus, where it is replicated and expressed. The details of the mechanism of transfection are not known. Both methods are employed to establish transiently expressed vectors while the calcium phosphate procedure is also used for permanently expressed foreign genes. Typically, 10–20% of the cells in culture can be transfected by these procedures.
DNA Elements Required for Expression of Vectors in Mammalian Cells
Expression of recombinant genes in mammalian cells requires the presence of DNA­controlling elements within the vector that are not necessary in the bacterial system. To be expressed in a eukaryotic cell the cloned gene is inserted in the vector in the proper orientation relative to control elements, including a promoter, polyadenylation signals, and an enhancer sequence. Expression may be improved by the inclusion of an intron. Some or all of these DNA elements may be present in the recombinant gene if whole genomic DNA is used for cloning. A particular cloned fragment generated by restriction endonuclease cleavage, however, may not contain the required controlling elements. A cDNA would not possess these required DNA elements. It is therefore necessary that the expression vector to be used in mammalian cells be constructed such that it contains all of the required controlling elements.
An expression vector can be constructed by insertion of required DNA­controlling elements into the vector by recombinant technologies. Enhancer and promoter elements, engineered into an expression vector, should be recognized by a broad spectrum of cells in culture for the greatest applicability of the vector. Controlling elements derived from viruses with a broad host range are used for this purpose and are usually derived from the papovavirus, simian virus 40 (SV40), Rous sarcoma virus, or the human cytomegalovirus.
The vector must replicate so as to increase the number of copies within each cell or to maintain copies in daughter cells. The vector therefore is constructed to contain DNA sequences that promote its replication in the eukaryotic cell. This DNA region is usually derived from a virus and is referred to as the origin of replication (Ori). Specific protein factors, encoded by genes engineered into the vector or previously introduced into the host genome, recognize and interact with the ori sequences to initiate DNA replication.
Transfected Eukaryotic Cells Can Be Selected by Utilizing Mutant Cells That Require Specific Nutrients
It is important to have a means of selectively growing the transfected cells since they often represent only 10–20% of the cell population. As was the case for the bacterial plasmid, a gene can be incorporated into the vector that encodes an enzyme that confers resistance to a drug or confers selective growth capability to the cells carrying the vector. Constructing vectors that express both a selectable marker and a foreign gene is difficult. Cotransfection circumvents this problem. Two different vectors are efficiently taken up by those cells capable of being transfected. In most cases greater than 90% of transfected cells carry both vectors, one with the selectable marker and the second carrying the gene of interest.
Two of the more commonly employed selectable markers are the thymidine kinase (tk) and the dihydrofolate reductase gene. The tk gene product, thymidine kinase, is expressed in most mammalian cells and participates in the salvage pathway for thymidine. Several mutant cell lines have been isolated that lack a functional thymidine kinase gene (tk–) and in growth medium containing hypoxanthine, aminopterin, and thymidine these cells will not survive. Only
Fly UP