Amplicon Express

Conway, J. E., Zolotukhin, S., Muzyczka, N., Hayward, G. S. and Byme, B. J. (1997). Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex vims type 1 amplicon expressing Rep and Cap. J. Virol. 71, 8780-8789. 

Lahr et al.59 Microdissected thyroid tissues PUREscript kit (BlOzym, Germany) Nested PCR, RET oncogene with amplicon size of 141-383 bp Expressed genes can be analyzed from routine FFPE tissue slides or pooled single cells. 

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [>600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

The tag sequence is amplified from plasmid pTA-His that provides a flexible linker and a double (His)g tag. The two amplicons can then be assembled and amplified by using an upstream T7 primer containing the promoter plus Kozak sequence and start codon for translation. A T-term/ for primer is used downstream to amplify across the tag and linker. The resulting construct contains the gene, the promoter, and the tag (Figure 6.26). This is key to the technology for the construct is then used for cell-free expression of the protein. 

FIGURE 4.3 Schematic representation comparing the full and deconstructed virus vector strategies, (a) TMV expression vector, (b) Provector system for rapid assembly of viral amplicons in planta. P promoter, TMV Pol TMV polymerase, MP movement protein, GOI gene of interest, CP capsid protein, T terminator, RS recombination site. (Adapted from Gleba et al. (2004). Curr. Opin. Plant Biol., 7, 182-188.)... 

Liu, Y., Rabinovitch, A., Suarez-Pinzon, W., Muhkeijee, B., Brownlee, M., Edelstein, D. etal. (1996) Expression of the bcl-2 gene from a defective E1SV-1 amplicon vector protects pancreatic beta-cells from apoptosis. Hum. Gene Ther., 7, 1719-1726. 

Hocknell PK, Wiley RD, Wang X, Evans TG, Bowers WJ, Hanke T, Federoff HJ, Dewhurst S (2002) Expression of human immunodeficiency virus type 1 gpl20 from herpes simplex vuus type 1-derived amplicons results in potent, specific, and durable cellular and humoral immune responses. J Virol 76 5565—5580. 

Bowers WJ, Howard DF, Federoff HJ (2000) Discordance between expression and genome transfer titering of HSV amplicon vectors Recommendation for standardized enumeration. Mol Ther 1 294-299. 

Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers. Gene Ther 8 111-120. 

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