Vectors/Delivery of DNA

Vectors

Plasmids:

A circular plasmid DNA can be cleaved with one enzyme and then joined to a linear chromosomal DNA fragment cut with the same enzyme using DNA ligase to form a recombinant plasmid DNA molecule. In one method called "tailing" a terminal deoxynucleotidyl transferase enzyme can be used to add short stretches of poly C to the 3' ends of molecule of interest (such as a cDNA or DNA) and short stretches of poly G to the 3'ends of the plasmid DNA which has been cut with a restriction enzyme that generates ds blunt ends. This way, the poly C ends of the dsmolecule of interest can be ligated with the poly G tail of the vector molecule. Among the advantages of plamsids are:

  • no size limitations

Disadvantages include:

  • low efficiency

Liposomes are vesicles composed of phospholipid bilayer membranes that can enclose various substances, such as DNA. Compared to naked DNA approaches, lipoplex can mediate inflammatory and/or immune reactions and possibly distinct toxicity profiles against normal tissue of the lipid components used to complex DNA. Compared to viral vectors, it can transfect various different cells without the need for interaction with specific receptors and possibility of multiple administration. However, transfection efficiency is generally low compared to viral vectors. Among the advantages are easy to produce, safe, less likely to produce immune response, no limitation on size. low efficiency

Viruses:

A DNA fragment can be ligated as above into a number of different viruses. Some common viruses that can be used as vectors are the following:

bacteriophage ג  (which can then be used to infect E. Coli)

retrovirus expression vector  such as the Moloney murine leukemia virus. When a retrovirus is used as a vector, most of the retroviral genes are removed so that the vector cannot produce viral particles. The genes left include a strong promoter region located at the 5' end of the viral genome called the long terminal repeat (LTR). The cloned DNA construct in the vector will integrate into the host DNA that codes for the necessary viral proteins (GAG, POL, ENV) Among the advantages of retroviruses as vectors are the following:

  • efficient transfer, high efficiency, stable expression for the life of the host cell

Disadvantages include:

  • carries small DNA sequences only, low transduction efficiency, integration with potential oncogenesis, poor in vivo delivery, risk of replication, infection requires efficient cell division and certain binding surface envelope protein only dividing cells (but lentiviruses, such as HIV, can infect non-dividing cells)

Episomal EBV or LANA expression vector goes into the nucleus and survives as an episome.

DNA virus (adenovirus) expression vector. can accommodate up to 7.0 kb of cloned DNA. The adenovirus has a deletion of the viral E1 proteins. It can infect a host cell that has DNA which produced viral E1 proteins which can then combine with the clone genes in the adenovirus. There is no integration here. Among the advantages of adenovirus are:

  • highly efficient transfer, targets nondividing cells and has broad host range, nontoxic to host cell, high transduction efficiency.

Among the disadvantages are:

  • possible host immune reaction, risk of replication, carries small DNA sequences, low potential oncogenesis, no integration, transient expression

Adeno-associated Virus

  • less likely to produce immune reactions, targets nondividing cells, efficient transfer, integrates into genome

  • small capapcity, immunogenic, not well studied, risk of replication

Herpes Simplex Virus

  • infects non-dividing cells and carry large regions of exogenous DNA. no size limitation

  • low efficiency and needs to intereact with cell surface receptors.

Commonly Used Vectors in Cloning:

The following expression vectors are commonly used in cloning:

  • T7-based expression vectors: are convenient expression vectors because they can be induced with IPTG which binds to the lac represssor changing its conformation so that it is unable to bind to the operon site whereupon expression can proceed.

Delivery of Vectors

Naked Physical Injection Transfection efficiency is usually low in tumor cells, however, the transfection efficiency and duration of expression is long for muscle cells.

Gene Gun: involves coating DNA with gold particles followed by gene gun injection. Only a few layers of epidermal cells can be transfected. As an immuno-approach (particularly cytokine genes), this approach can be successful.

Electroinjection: involves physical injection of DNA in saline followed by electroporation. Transfection efficiency is generally higher than injecting DNA in saline alone.

 

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