15-01-2014, 01:16 PM
Artificial Chromosomes
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INTRODUCTION
Artificial chromosomes are popular because they carry large amounts of genetic material. The yeast artificial chromosome (YAC) is one of the most widely used. YACs are stretches of DNA that contain all the elements required to propagate a chromosome in yeast: a replication origin, the centromere required to segregate chromatids into daughter cells, and two telomeres to mark the ends of the chromosome. They will also have restriction enzyme sites and genetic markers so that they can be traced and selected. Cleavage of a YAC with the proper restriction enzyme such as SmaI will open it up and allow the insertion of a piece of foreign DNA between the centromere and a telomere. In this way YACs containing DNA fragments between 100 and 2,000 kilobases in size can be placed in Saccharomyces cerevisiae cells and will be replicated along with the true chromosomes. The bacterial artificial chromosome (BAC) is an increasingly popular alternativecloning vector. BACs are cloning vectors based on the E. coli F-factor plasmid. They contain appropriate restriction enzyme sites and a marker such as chloramphenicol resistance. The modified plasmid is cleaved at a restriction site, and a foreign DNA fragment up to 300 kilobases in length is attached using DNA ligase. The BAC is reproduced in E. coli after insertion by electroporation.This vector is easy to reproduce and manipulate and does not undergo recombination as readily as YACs (which means that the insert is less likely to be rearranged). Because theycan carry such large fragments of DNA, artificial chromosomes have been particularly useful in genome sequencing.
Recombinant antibodies
Monoclonal antibodies (mAbs) are ubiquitous in biomedical research and medicine. They are used to fight, diagnose and research disease and to develop and test new drugs. While the mouse ascites method of mAb production is widely discouraged due to the substantial pain and distress involved and the equivocally named in vitro method has become standard, both methods raise serious animal welfare concerns. Fortunately, an alternative to animal-based mAbs exists. Synthetic antibodies called recombinant antibodies (rAbs) can be created using antibody genes made in a laboratory or taken from human cells, completely eliminating animals from the antibody-production process. rAbs can be used in all applications in which traditional mAbs are used and have inherent advantages over their animal-derived counterparts as well. The following review will discuss the production of rAbs, their advantages, and some impediments to their use.
Production of Recombinant Antibodies
The advent of recombinant DNA technology10,11,12 laid the foundation for recombinant antibody production by enabling the combination of genetic material from two or more sources. In 1990 John McCafferty and colleagues demonstrated that antibody fragments could be displayed on viruses that infect bacteria, called bacteriophages or phages, by introducing antibody DNA into phage genomes via vectors.13 Researchers then created libraries of antibody genes to display on phages and developed methods to successfully isolate individual antibodies from the large phage-displayed libraries. This method has become known as antibody phage display. Of the 22 FDA approved therapeutic monoclonal antibodies currently on the market, one was created through non-animal antibody phage display technology. Of the previously mentioned antibody initiatives, both the German Antibody Factory and the European ProteomeBinders consortium create non-animal recombinant antibodies using antibody phage display. Since 1990, researchers have expanded antibody display technology to microorganisms other than phage. Koder and Wittrup, for example, documented the display of antibody fragments on Saccharomyces cerevisiae yeast in 1997.The production of non-animal recombinant antibodies can be broken down into five steps: (1) creation of an antibody gene library; (2) display of the library on phage coats or cell surfaces (3) isolation of antibodies against an antigen of interest; (4) modification of the isolated antibodies and (5) scaled up production of selected antibodies in a cell culture expression system.
Maturation and Structure Conversion:
Upon repeated exposure to an antigen, an animal will create progressively stronger antibodies against it. Antibodies generated during the primary response to an antigen are mutated and a secondary response is mounted. This process, called affinity maturation, may repeat itself several times until strong antibodies that can neutralize an antigen or eliminate it from the body are generated. Traditional monoclonal antibody technology takes advantage of an animal's natural affinity maturation process by immunizing an animal several times with the same antigen over a period of weeks. Recombinant antibody technology requires that researchers carry out the affinity maturation process themselves with advanced molecular biology techniques.Affinity matured antibody fragments selected from an antibody library may also need to be converted to divalent or full-length antibodies to increase their utility in specific applications.