Cloning+Vector

=__Cloning Vector __=


 * Basic Description **

DNA cloning results in the generation of identical copies of a nucleic acid segment of interest. Researchers commonly replicate genes of interest in order to further understand their expression. The process involves several steps which have become fairly fast and efficient due to the emergence of new biotechnology. The replication begins with the insertion of the fragment of interest into a purified DNA genome of a self-replicating genetic element (Alberts, Johnson, Lewis, Raff, Roberts, & Walter, 2008). This is done using either genetic elements from bacteria or viruses. Using viruses, a fragment is inserted into their DNA then relocated into a bacteria cell which allows for the replication of the viral DNA along with the inserted fragment. This method is capable of replicating one genetic element up to ten trillion copies over the course of a 24 hour period. The bacterial cloning vectors are small circular molecules of double-stranded DNA derived from larger vectors of bacterial origin. The smaller DNA elements are first separated through centrifugation from larger DNA chromosomes. Then the DNA circles are cut using endonucleases to form linear DNA molecules. The target DNA for replication is also cut using the same endonucleases so that they have the same cohesive ends, allowing for them to be annealed. The ends of the combined linear DNA molecule are then connected to form a circular molecules using DNA ligase (Alberts, Johnson, Lewis, Raff, Roberts, & Walter, 2008).

After formation of the vector a species of bacterial cells are chosen to be transfected. The host cells are made permeable to DNA and then the cloning vector is capable of penetration. As the host cell replicates every 20-30 minutes depending on the species, large amounts of the DNA of interest are in turn replicated. Many of these cloning vectors use markers such as fluorescence or antibiotic resistance in order for researchers to detect and isolate what cells were able to uptake and replicate the DNA. The most commonly used vectors are Bacterial Artificial Chromosomes. These vectors are a derivative of the naturally occurring F plasmid of E.coli. The positives of this specific element are that it is capable of cloning DNA fragments of 300,000 to 1 million base pairs. These vectors have been shown to occur in low numbers in their host cells and in turn maintain the integrity of the fragment (Alberts, Johnson, Lewis, Raff, Roberts, & Walter, 2008).

The benefits of using this specific type of plasmid over others is the size of the DNA molecule which you can clone. Since this vector is Eukaryotic in nature the size is much larger than those from smaller microbes like E.coli k-12. The three letter regions labeled: TEL, ORI, and CEN are seen as the sequences for the telomere, origin of replication, and centromere (Alberts et al., 2008).


 * Figure 1: Yeast artificial chromosome (YAC) **


 * Purpose of Technique **

This technique serves many purposes as it shows great capability in the creation of DNA libraries. This allows researchers to study the macromolecule roles in cellular function, development, maintenance. Creation of genetic libraries aid in displaying variance among different species as well as evolutionary relationships. It also proves pertinent in medicine as understanding how mutations and variability in expression cause diseases allows for the development of possible treatments (Alberts, Johnson, Lewis, Raff, Roberts, & Walter, 2008).


 * Origins and History **

The origins of these techniques can be traced back to several individuals around the time period of the 1970’s. During this time genomic studies were limited due to lack of isolation and replication techniques for use in highly developed organisms. In 1972, David A. Jackson, Robert H. Symons, and Paul Berg, published the article, “Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli”, in the journal, Proceedings of the National Academy of Sciences of the United States of America. This article established the foundation for molecular cloning as they created a reproducible method for insertion of DNA into viral genomes allowing replication. In 1973, Stanley N. Cohen, Annie C. Y. Chang, Herbert W. Boyer, and Robert B. Helling published the article,” Construction Biologically Functional Bacterial Plasmids In Vitro”, in the journal, Proceedings of the National Academy of Sciences of the United States of America. Their work provided methodology for the synthesis of bacterial plasmids for use in genetic element cloning. In 1975, Daniel Nathans and Hamilton O. Smith published the article, “Restriction Endonucleases in the Analysis and Restructuring of DNA Molecules”, in the journal, Annual Review of Biochemistry. The application of endonucleases to molecular cloning greatly expanded the possibilities of vector and target DNA combinations.


 * Recent Research **

Many recent articles involve the use of genomic cloning technology for various applications. In 2005, Anthony L. Contento, Yan Xiong, and Diane C. Bassham, published the article, “Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein” in The Plant Journal. Their use of this technique allowed for replication of the protein of interest fusion protein, GFP-AtATG8e. They performed the replication using modified pJ4GFP-XB vectors. They then used this protein as a fluorescent marker as it binded to several proteins associated with autophagosomes of Arabidopsis. They were able to visualize these membrane structures using confocal microscopy, demonstrating that ATG8 homologs could be used as autophagosome markers in Arabidopsis.

In 2012, the article, “Gene silencing of IL-12 in dendritic cells inhibits autoimmune arthritis” was published in The Journal of Translational Medicine. The researcher’s cloned IL-12 shRNA element using the Psilencer 3.1 vector for studies involving suppression of autoimmune arthritis. After replication, the genetic element was inserted into bone marrow derived dendritic cells. These cells containing the silenced RNA molecule were inserted into mice where the autoimmune arthritis had been induced. Their studies demonstrated a DNA-directed RNA interference which allowed for the suppression of the T-cells involved and in turn the immune response.

In 2012, the article, “A new cloning system based on the Oprl lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations”, was published in the Journal of Biotechnology. The authors applied molecular cloning to structurally modify antigens during replication. They were able to generate further evidence on how human immune response is based on recognition of viral and bacterial essential components like structural elements. They cloned antigens associated with viruses such as African swine fever and HIV using cloning vectors which were modified with certain ligands and lipoproteins that allowed the human immune response to not only act faster but more efficiently in its’ response.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2008). Molecular Biology of THE CELL. New York : Garland Science, Taylor & Francis Group.
 * References **