Vectors for Gene Transformation

Sathiyaraj Srinivasan

2006315223

Viral vector Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage lambda to infect monkey kidney cells maintained in culture.

Key properties of viral vector viral vectors are tailored to their specific applications but generally share a few key properties.

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Safety. Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions. Low toxicity. The viral vector should have a minimal effect on the physiology of the cell it infects. Stability. Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design. Cell type specificity. Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell.

Applications

Basic research Viral vectors were originally developed as an alternative to transfection of naked DNA for molecular genetic experiments. Compared to traditional methods such as calcium phosphate precipitation, transduction can ensure that nearly 100% of cells are infected without severely affecting cell viability. Furthermore, some viruses integrate into the cell genome facilitating stable expression. However, transfection is still the method of choice for many applications as construction of a viral vector is a much more laborious process. Protein coding genes can be expressed using viral vectors, commonly to study the function of the particular protein. Viral vectors, especially retroviruses, stably expressing marker genes such as GFP are widely used to permanently label cells to track them and their progeny, for example in xenotransplantation experiments, when cells infected in vitro are implanted into a host animal. Genes inserted into the vector can encode shRNAs and siRNAs used to efficiently block or silence production of a specific protein. Such knock-down experiments are much quicker and cheaper to carry out than gene knockout. But as the silencing is sometimes non-specific and has offtarget effects on other genes, it provides less reliable results. Gene therapy In the future gene therapy may provide a way to cure genetic disorders, such as severe combined immunodeficiency or cystic fibrosis. Several gene therapy trials have used viruses to deliver 'good' genes to the cells of the patient's body. There have been a huge number of laboratory successes with gene therapy. However, several problems of viral gene therapy must be overcome before it gains widespread use. Immune response to viruses not only impedes the delivery of genes to target cells but can cause severe complications for the patient. In one of the early gene therapy trials in 1999 this led to the death of Jesse Gelsinger, who was treated using an adenoviral vector. Some viral vectors, for instance lentiviruses, insert their genomes at a seemingly random location on one of the host chromosomes, which can disturb the function of cellular genes and lead to cancer. In a severe combined immunodeficiency retroviral gene therapy trial conducted in 2002, two of the patients developed leukemia as a consequence of the treatment.[3] Adeno-associated virus-based

vectors are much safer in this respect as they always integrate at the same site in the human genome.

Vaccines Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as DNA vaccines. Tlymphocytes recognize cells infected with intracellular parasites based on the foreign proteins produced within the cell. T cell immunity is crucial for protection against viral infections and such diseases as malaria. A viral vaccine induces expression of pathogen proteins within host cells similarly to the Sabin Polio vaccine and other attenuated vaccines. However, since viral vaccines contain only a small fraction of pathogen genes, they are much safer and sporadic infection by the pathogen is impossible. Adenoviruses are being actively developed as vaccines. Types of viral vectors Retroviruses Retroviruses are the one of mainstays of current gene therapy approaches. The recombinant retroviruses such as the Moloney murine leukemia virus have the ability to integrate into the host genome in a stable fashion. They contain a reverse transcriptase which allows integration into the host genome. They have been used in a number of FDA-approved clinical trials such as the SCID-X1 trial.[4] The primary drawback to use of retroviruses such as the Moloney retrovirus involves the requirement for cells to be actively dividing for transduction. As a result, cells such as neurons are very resistant to infection and transduction by retroviruses. There is a concern for insertional mutagensis due to the integration into the host genome which can lead to cancer or leukemia. Lentiviruses

Packaging and infection by a lentiviral vector

. Lentiviruses are a subclass of Retroviruses. They are widely adapted as vectors thanks to their ability integrate into the genome of non-dividing as well as dividing cells. The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme. The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. The site of integration is unpredictable, which can pose a problem. The provirus can disturb the function of cellular genes and lead to activation of oncogenes promoting the development of cancer, which raises concerns for possible applications of lentiviruses in gene therapy. For safety reasons lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several plasmids are transfected into a socalled packaging cell line, commonly HEK 293. One or more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce the singlestranded RNA viral genome and is marked by the presence of the ψ (psi) sequence. This sequence is used to package the genome into the virion. Adenoviruses As opposed to lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. This limits their use in basic research, although adenoviral vectors are occasionally used in in vitro experiments. Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with

potentially dangerous consequences. To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity. Nanoengineered Substances Substances such as Ormosil have been successfully used as a DNA vector.

Plasmid A 'plasmid' is a DNA molecule separate from the chromosomal DNA and capable of autonomous replication. In many cases, It is typically circular and double-stranded. It usually occurs in bacteria, and is sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae). The size of plasmids varies from 1 to over 400 kilobase pairs (kbp). There may be one copy, for large plasmids, to hundreds of copies of the same plasmid in a single cell, or even thousands of copies, for certain artificial plasmids selected for high copy number (such as the pUC series of plasmids). Plasmids can be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer. The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952. Antibiotic resistance

Figure 2: Illustration of a plasmid with antibiotic resistances. 1 & 2 Genes that code for resistance. 3 Ori.

Plasmids often contain genes or gene cassettes that confer a selective advantage to the bacterium harboring them, such as the ability to make the bacterium antibiotic resistant. Every plasmid contains at least one DNA sequence that serves as an origin of replication, or ori (a starting point for DNA replication), which enables the plasmid DNA to be duplicated independently from the chromosomal DNA (Figure 2). The plasmids of most bacteria are circular, like the plasmid depicted in Figure 2, but linear plasmids are also known, which superficially resemble the chromosomes of most eukaryotes. Episomes An episome is a plasmid that can be added, without integration, to the chromosomal DNA of the host organism (Fig. 3). In this situation, it can stay intact for a long time, be either diluted out or be duplicated with every cell division of the host, and become a basic part of its genetic makeup. The term is no longer commonly used for plasmids, since it is now clear that a region of homology with the chromosome such as a transposon will make a plasmid into an episome. In mammalian systems, the term episome refers to a circular DNA (such as a viral genome) that is maintained by noncovalent tethering to the host cell chromosome. In detail, we distinguish:

Minichromosomes (MCs) that contain all elements for replication and segregation (telomeres, centromeres); MCs are usually obtained by chromosome engineering (e.g. telomere-seeding, the use of site-specific recombinases and/or the recombinase-mediated cassette exchange concept) Plasmids and Minicircles that stay extra-chromosomal (episomal) in nonor barely-dividing tissues (note: ´minicircles´ are plasmids devoid of prokaryotic sequence parts and thereby poor in inactivating CpG tracts)

Replicating minicircles, i.e. plasmid derivatives that can recruit all components necessary for autonomous replication from the host cell. They replicate once per cell cycle and do not undergo epigenetic inactivation. In contrast to viral circular episomes (SV40, BPV, EBV, HSV) no viral components (proteins) are required for autonomous relpication.

Figure 3: Comparison of non-integrating plasmids (top) and episomes (bottom). Top: Chromosomal DNA and plasmids replicated separately. Bottom: Chromosomal DNA with integrated plasmids replicate as a single chromosome. Vectors Plasmids used in genetic engineering are called vectors. They are used to transfer genes from one organism to another and typically contain a genetic marker conferring a phenotype that can be selected for or against. Most also contain a polylinker or multiple cloning site (MCS), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. See Applications below.[citation needed]

Types

Figure 4: Overview of Bacterial conjugation One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-genes, which perform the complex

process of conjugation, the sexual transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitise' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases. It is possible for plasmids of different types to coexist in a single cell. Seven different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups. Another way to classify plasmids is by function. There are five main classes:
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Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation. Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood. Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria. Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid. Virulence plasmids, which turn the bacterium into a pathogen.

Plasmids can belong to more than one of these functional groups. Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such singlecopy plasmids have systems which attempt to actively distribute a copy to both daughter cells. Some plasmids include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid

R1 in Escherichia coli.[2] They produce both a long-lived poison and a shortlived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell. Applications Plasmids serve as important tools in genetics and biochemistry labs, where they are commonly used to multiply (make many copies of) or express particular genes.[3] Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid which contains genes that make cells resistant to particular antibiotics. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics. Plasmid DNA extraction As alluded to above, plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often need to be isolated.

There are several methods to isolate plasmid DNA from bacteria, the archetypes of which are the miniprep and the maxiprep.[3] The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques. In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA. In recent times many commercial kits have been created to perform plasmid extraction at various scales, purity and levels of automation. Commercial services can prepare plasmid DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram quantities (early 2007). Conformations Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
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"Nicked Open-Circular" DNA has one strand cut. "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself. "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself. "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself.

"Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation. You can model this by twisting a badly frayed extension cord and then plugging it into itself.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage. At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments. Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA Cloning vector

The pGEX-3x plasmid is a popular cloning vector.

A cloning vector is a small DNA vehicle that carries a foreign DNA fragment. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Plasmids and bacteriophages (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Common Features Most commercial cloning vectors have a couple of key features that have made their use in molecular biology so widespread. Usually, the main purpose of cloning vector is the controlled expression of a particular gene inside a convenient host organism (eg. E. coli). Control of expression can be very important; it is usually desirable to insert the target DNA into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters, lac promoters (bla promoter) and cauliflower mosaic virus's 35s promoter (for plant vectors). To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites engineered out of them and a multiple cloning site (MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as antibiotic resistance [eg. beta-lactamase (see figure)] is often included in the vector to identify positively transformed cells. All inserted DNA (plasmids etc.) need an origin of replication (ORI; not shown in figure). High stringency ORIs are preferable for cloning vectors. Some other possible features of cloning vectors are: vir genes for plant transformation, intergrase sites for chromosomal insertion, lac Z alpha fragment for blue-white selection, and/or in-frame genes attached to the MCS for recombinant proteins [eg. Green fluorescent protein (GFP) or glutathione Stransferase (see figure)]. Screening: blue and white selection

General purpose vectors such as pUC19 usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used beta-galactosidase can be detected by its ability to change the substrate X-gal (5 bromo-4-chloro-3-indolyl-beta-d-galactoside) from colourless to blue. Cloning a fragment of DNA within the vector based gene encoding the beta galactosidase prevents the formation of an active beta galactosidase. If x gal is included in the selective agar plates, transformant colonies are blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.

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