The Basic Properties of a Cloning Vector
Cloning vectors were developed in the early 1970s from naturally occurring DNA molecules found in some cells of the bacteria
Escherichia coli
(E. coli). These replicating molecules, called plasmids, were first used by the American scientists Stanley Cohen
and Herbert Boyer
as vehicles, or vectors, to replicate other pieces of DNA (insert DNA) that were joined to them. Thus the first two essential features of cloning vectors are their ability to replicate in an appropriate host cell and their ability to join to foreign DNA sequences to make recombinant molecules. Plasmid replication requires host-cell-specified enzymes, such as DNA polymerases that act at a plasmid sequence called the “origin of replication.” Insert DNA is joined (ligated) to plasmid DNA through the use of two kinds of enzymes: restriction enzymes and DNA ligases. The plasmid DNA sequence must have unique sites for restriction enzymes to cut. Cutting the double-stranded circular DNA at more than one site would cut the plasmid into pieces and would separate important functional parts from one another. However, when a restriction enzyme cuts the circular plasmid at one unique site, it converts it to a linear molecule. Linear insert DNA molecules, produced by cutting DNA with the same restriction enzyme as was used to cut the plasmid vector, can be joined to cut plasmid molecules using the enzyme DNA ligase. This catalyzes the covalent joining of the insert DNA and plasmid DNA ends to create a circular, recombinant plasmid molecule. Most cloning vectors have been designed to have many unique restriction enzyme cutting sites all in one stretch of the vector sequence. This part of the vector is referred to as the multiple cloning site.
In addition to an origin of replication and a multiple cloning site, most vectors have a third element: a selective marker. In order for the vector to replicate, it must be present inside an appropriate host cell. Introducing the vector into cells is often a very inefficient process. Therefore, it is very useful to be able to select, from a large population of host cells, those rare cells that have taken up a vector. This is the role of the selectable marker. The selectable marker is usually a gene that encodes resistance to an antibiotic to which the host is normally sensitive. For example, if a plasmid vector has a gene that encodes resistance to the antibiotic ampicillin, only those E. coli cells that harbor a plasmid will be able to grow on media containing ampicillin.
Many vectors have an additional selective marker that is rendered inactive when a plasmid is recombinant. A commonly used marker gene of this kind is the lacZ gene, which encodes the enzyme beta-galactosidase. This enzyme breaks the disaccharide lactose into two monosaccharides. The pUC plasmid vector has a copy of the lacZ gene which has been carefully engineered to contain a multiple cloning site within it, while maintaining the functionality of the expressed enzyme. When a DNA fragment is inserted into the multiple cloning site, the lacZ gene is no longer capable of making functional beta-galactosidase. This loss of function can be detected by putting X-gal into the growth media. X-gal has a structure similar to lactose but cannot be broken down by beta-galactosidase. Rather, beta-galactosidase modifies X-gal and produces a blue color. Thus, colonies of the bacterium E. coli containing recombinant plasmids will be normal colored, whereas those that have normal,
nonrecombinant plasmids will be blue. Typical selection media then contain ampicillin and X-gal. The ampicillin only allows E. coli that contain a plasmid to grow, and the X-gal identifies which colonies have recombinant plasmids.
There are a number of procedures for introducing the plasmid vector into the host cell. Transformation is a procedure in which the host cells are chemically treated so that they will allow small DNA molecules to pass through the cell membrane. Electroporation is a procedure that uses an electric field to create pores in the host cell membrane to let small DNA molecules pass through.
Viruses and Cloning Vectors
In addition to plasmid cloning vectors, some bacteriophages (or phages) have been modified to serve as cloning vectors. Bacteriophages, like other viruses, are infectious agents that are made of a genome, either DNA or RNA, that is surrounded by a protective protein coat. Phage vectors are used similarly to the way plasmid vectors are used. The vector and insert DNAs are cut by restriction enzymes so that they subsequently can be joined by DNA ligase. The newly formed recombinant DNA molecules must enter an appropriate host cell to replicate. In order to introduce the phage DNA into cells, a whole phage particle must be built. This is referred to as “packaging” the DNA. The protein elements of the phage are mixed with the recombinant phage DNA and packaging enzymes to create an infectious phage particle. Appropriate host cells are then infected with it. The infected cells then make many copies of each recombinant molecule, along with the proteins needed to make a completed phage particle. In many cases, the final step of viral infection is the lysis of the host cell. This releases the mature phage particles to infect nearby host cells. Phage vectors have two advantages relative to plasmid vectors: First, viral delivery of recombinant DNA to host cells is much more efficient than the transformation or electroporation procedures used to introduce plasmid DNA into host cells, and second, phage vectors can be used to clone larger fragments of insert DNA.
Viruses that infect cells other than bacteria have been modified to serve as cloning vectors. This permits cloning experiments using many different kinds of host cells, including human cells. Viral vectors, just like the natural viruses from which they are derived, have specific host and tissue ranges. A particular viral vector will be limited for use in specific species and cell types. The fundamental practice of all virally based cloning vectors involves the covalent joining of the insert DNA to the viral DNA to make a recombinant DNA molecule, introduction of the recombinant DNA into the appropriate host cell, and then propagation of the vector through the natural mechanism of viral replication. There are two fundamentally different ways that viruses propagate in cells. Many viruses, such as the phages already described, enter the host cell and subvert the cell’s biosynthetic machinery to its own reproduction, which ultimately leads to lysis and thereby kills the host cell as the progeny viruses are released. The second viral life strategy is to enter the host cell and integrate the viral DNA into the host cell chromosome so that the virus replicates along with the host DNA. Such integrating viruses can be stably maintained in the host cell for long periods. The retroviruses, of which the human immunodeficiency virus (HIV) is an example, are a group of integrating viruses that are potentially useful vectors for certain gene therapy applications. Using cloning vectors and host cells other than bacteria allows scientists to produce some proteins that bacteria cannot properly make, permits experiments to determine the function of cloned genes, and is important for the development of gene therapy.
Expression Vectors
Expression vectors are cloning vectors designed to express the gene contained in the recombinant vector. In order to accomplish this, they must also provide the appropriate regulatory signals for the transcription and translation of the foreign gene. Regulatory sequences, which direct the cellular transcription machinery, are very different in bacteria and higher organisms. Thus, unless the vector provides the appropriate host regulatory sequences, foreign genes will not normally be expressed.
Expression vectors make it possible to produce proteins encoded by eukaryotic genes (that is, genes from higher organisms) in bacterial cells. Furthermore, producing proteins in this way often results in higher production rates than in the cells from which the gene was obtained. This technology not only is of immense benefit to scientists who study proteins but also is used by industry (particularly the pharmaceutical industry) to make valuable proteins. Proteins such as human insulin, growth hormone, and clotting factors that are difficult and extremely expensive to isolate from their natural sources are readily available because they can be produced much more cheaply in bacteria. An added benefit of expression vectors is that actual human proteins are produced by bacteria and therefore do not provoke allergic reactions as frequently as insulin that is isolated from other species.
Artificial Chromosomes
In 1987, a new type of cloning vector was developed by David Burke, Maynard Olson, and their colleagues. These new vectors, artificial chromosomes, filled the need created by the Human Genome Project (HGP) to clone very large insert DNAs (hundreds of thousands to millions of base pairs in length). One of the goals of the HGP—to map and ultimately sequence all the chromosomes of humans, as well as a number of other “model” organisms’ genomic sequences—required a vector capable of propagating much larger DNA fragments than plasmid or phage vectors could propagate. The first artificial chromosome vector was developed in the yeast Saccharomyces cerevisiae. All the critical DNA sequence elements of a yeast chromosome were identified and isolated, and these were put together to create a yeast artificial chromosome (YAC). The elements of a YAC vector are an origin of replication, a centromere, telomeres, and a selectable marker suitable for yeast cells. A yeast origin of replication (similar to the origin of replication of bacterial plasmids) is a short DNA sequence that the host’s replicative enzymes, such as DNA polymerase, recognize as a site to initiate DNA replication. In addition to replicating, the new copies of a chromosome must be faithfully partitioned into daughter cells during mitosis. The centromere sequence mediates the partitioning of the chromosomes during cell division because it serves as the site of attachment for the spindle fibers in mitosis. Telomeres are the DNA sequences at the ends of chromosomes. They are required to prevent degradation of the chromosome and for accurate replication of DNA at the ends of chromosomes.
YACs are used much as plasmid vectors are. Very large insert DNAs are joined to the YAC vector, and the recombinant molecules are introduced into host yeast cells in which the artificial chromosome is replicated just as the host’s natural chromosomes are. YAC cloning technology allows very large chromosomes to be subdivided into a manageable number of pieces that can be organized (mapped) and studied. YACs also provide the opportunity to study DNA sequences that interact over very long distances. Since the development of YACs, artificial chromosome vectors for a number of different host cells have been created.
Impact and Applications
Cloning vectors are one of the key tools of recombinant DNA technology. Cloning vectors make it possible to isolate particular DNA sequences from an organism and make many identical copies of this one sequence in order to study the structure and function of that sequence apart from all other DNA sequences. Until the development of the polymerase chain reaction (PCR), cloning vectors and their host cells were the only means to collect many copies of one particular DNA sequence. For long DNA sequences (those over approximately ten thousand base pairs), cloning vectors are still the only means to do this.
Gene therapy is a new approach to treating and perhaps curing genetic disease. Many common diseases are the result of defective genes. Gene therapy aims to replace or supplement the defective gene with a normal, therapeutic gene. One of the difficulties faced in gene therapy is the delivery of the therapeutic gene to the appropriate cells. Viruses have evolved to enter cells, sometimes only a very specific subset of cells, and deliver their DNA or RNA genome into the cell for expression. Thus viruses make attractive vectors for gene therapy. An ideal vector for gene therapy would replace viral genes associated with pathogenesis with therapeutic genes; the viral vector would then target the therapeutic genes to just the right cells. One of the concerns related to the use of viral vectors for gene therapy is the random nature of the viral insertion into the target cell’s chromosomes. Insertion of the vector DNA into or near certain genes associated with increased risk of cancer could theoretically
alter their normal expression and induce tumor formation.
Plasmid DNA vectors encoding immunogenic proteins from pathogenic organisms are being tested for use as vaccines. DNA immunization offers several potential advantages over traditional vaccine strategies in terms of safety, stability, and effectiveness. Genes from disease-causing organisms are cloned into plasmid expression vectors that provide the regulatory signals for efficient protein production in humans. The plasmid DNA is inoculated intramuscularly or intradermally, and the muscle or skin cells take up some of the plasmid DNA and express the immunogenic proteins. The immune system then generates a protective immune response. There are two traditional vaccination strategies: One uses live, attenuated pathogenic organisms, and the other uses killed organisms. The disadvantage of the former
is that, in rare cases, the live vaccine can cause disease. The disadvantage of the latter strategy is that the killed organism does not enter the patient’s cells and make proteins like the normal pathogen. Therefore, one part of the immune response, the cell-mediated response, is usually not activated, and the protection is not as good. In DNA immunization, the plasmids enter the patient’s cells, and the immunogenic proteins produced there result in a complete immune response. At the same time, there is no chance that DNA immunization will cause disease, because the plasmid vector does not carry all of the disease-causing organism’s genes.
Key terms
bacteriophage
:
a virus that infects bacterial cells, often simply called a phage
foreign DNA
:
DNA taken from a source other than the host cell that is joined to the DNA of the cloning vector; also known as insert DNA
plasmid
:
a small, circular DNA molecule that replicates independently of the host cell chromosome
recombinant DNA molecule
:
a molecule of DNA created by joining DNA molecules from different sources, most often vector DNA joined to insert DNA
restriction enzyme
:
an enzyme capable of cutting DNA at specific base pair sequences, produced by a variety of bacteria as a protection against bacteriophage infection
Bibliography
Anderson, W. French. “Gene Therapy.” Scientific American 273.3 (1995): 124. Print.
Brown, T. A. Gene Cloning and DNA Analysis: An Introduction. 6th ed. Hoboken: Wiley, 2010. Print.
Brown, T. A. “Vectors for Gene Cloning: Plasmids and Bacteriophages.” Gene Cloning and DNA Analysis: An Introduction. 5th ed. Malden: Blackwell, 2006. Print.
Cohen, Philip. “Creators of the Forty-Seventh Chromosome.” New Scientist 11 Nov. 1995: 34. Print.
Friedmann, Theodore. “Overcoming the Obstacles to Gene Therapy.” Scientific American 276.6 (1997): 95–101. Print.
Hassett, Daniel E., and J. Lindsay Whitton. “DNA Immunization.” Trends in Microbiology 4.8 (1996): 307–12. Print.
Jones, P., and D. Ramji. Vectors: Cloning Applications and Essential Techniques. New York: Wiley, 1998. Print.
Kouprina, Natalya, William C. Earnshaw, Hiroshi Matsumoto, and Vladimir Larionov. “A New Generation of Human Artificial Chromosomes for Functional Genomics and Gene Therapy.” Cellular and Molecular Life Sciences 70.7 (2013): 1135–48. Print.
Krebs, Jocelyn E., Elliott S. Goldstein, Stephen T. Kilpatrick, and Benjamin Lewin. Lewin's Essential Genes. 3rd ed. Burlington: Jones, 2013. Print.
Lodge, Julia, Peter A. Lund, and Steve Minchin. Gene Cloning: Principles and Applications. New York: Taylor, 2007. Print.
Lu, Quinn, and Michael P. Weiner, eds. Cloning and Expression Vectors for Gene Function Analysis. Natick: Eaton, 2001. Print.
Watson, James D., et al. Recombinant DNA—Genes and Genomes: A Short Course. 3d ed. New York: Freeman, 2007. Print.
