(a region containing multiple restriction enzyme sites into which an insert can be installed or removed), selection markers, and transcriptional promoters. The passenger DNA must integrate into the host cell's DNA or be carried into the cell as part of a biologically active molecule that can replicate independently. If this result is not achieved, the inserted gene will not be successfully transcribed. The most commonly used biological agent for transporting genes into bacterial and yeast cells is the plasmid, such as the e. coli bacterial plasmid pBR322. A plasmid is a small, double-stranded, closed circular extrachromosomal DNA molecule. This plasmid contains 4,361 bp and can transport relatively small amounts of DNA. Plasmids occur in many species of bacteria and yeasts. Sometimes, plasmids carry their own genes (e.g., the highly transmissible genes for antibiotic resistance in some bacterial species). An important feature of a plasmid is that it has an origin of replication (ori) site that allows it to multiply independently of a host cell's DNA. Although there can be more than one copy of a plasmid in a cell, the copy number is controlled by the plasmid itself.

Another type of cloning vector is the bacteriophage (Fig. 4.7). Bacteriophage A (lambda) possesses a genome of approximately 4.9 X 105 bp and can package large amounts of genetic material without affecting the infec-tivity of the phage. A large DNA library can be created, packaged in bacteriophage A , and, when the virus infects, inserted into cells. Hybridization is then detected by screening with DNA probes.87 In addition, there are special vectors called phagemids, vaccinia and adenovirus for cloning into mammalian cells, and yeast artificial chromosomes (YACs) that facilitate cloning in yeasts.88 Differences among these vectors concern the size of the insert that they will accept, the methods used in the selection of the clones, and the procedures for propagation.

Once the passenger DNA has been created and the plasmid vector cut (both with the same restriction enzyme), the insert is ligated into the plasmid along with a promoter (a short DNA sequence that enhances the transcription of the adjacent gene). Often, a gene imparting antibiotic resistance linked to the desired gene is inserted as a selection tool. The idea behind this is that if the gene is inserted in the proper location, the bacterial cell will grow on a medium containing the antibiotic. Bacteria that do not contain the resistance gene and, hence, lack the required gene will not grow. This makes the task of screening for integration of the desired gene easier. After the molecule is ligated, the vector is finally an rDNA molecule that can be inserted into a host cell.

Host cells can be bacteria (e.g., e. coli), eukaryotic yeast (Saccharomyces cerevisiae), or mammalian cell lines, including Chinese hamster ovary (CHO), African green monkey kidney (VERO), and baby hamster kidney (BHK). It is easy to grow high concentrations of bacteria and yeast cells in fermenters to yield high protein concentrations. Mammalian cell culture systems typically give poorer protein yields, but sometimes this is acceptable, especially when the product demands the key posttranslational modifications that do not occur in bacteria. Host cells containing the vector are grown in small-scale cultures and screened for the desired gene.89 When the clone providing the best protein yield is located, the organism is grown under carefully controlled conditions and used to inoculate pilot-scale fermentations. Parameters such as production medium composition, pH, aeration, agitation, and temperature are investigated at this stage to optimize the fermentation. The host cells divide, and the plasmids in them replicate, producing the desired "new" protein. The fermentation is scaled up into larger bioreactors for large-scale isolation of the recombinant protein. Obviously, the cultures secrete

Figure 4.7 • Types of cloning vectors: a bacteriophage and a plasmid.

their own natural proteins along with the cloned protein. Purification steps are required before the recombinant protein is suitable for testing as a new, genetically engineered pharmaceutical agent. Once the host cell line expressing the recombinant gene is isolated, it is essential to maintain selection pressure on it so that it does not spontaneously lose the plasmid. Typically, this pressure is applied by maintaining the cells on medium containing an antibiotic to which they bear a resistance gene.

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