Reverse genetic approach

PrPc has a predominantly a-helical conformation 796 Reverse genetics approaches to studying PrPc 797 The function of PrPc remains unknown 797 PrP knockout mice have subtle abnormalities of synaptic transmission 797... 

Reverse genetic approaches start with a sequence and attempt to identify a function for the gene. This can be done by using PCR-based screens on DNA from insertionally mutagenized populations, ectopic misexpression of the gene, and expression studies, e.g., with RT-PCR or microarray analyses. 

Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54 519-546 Dixon RA, Reddy MSS (2003) Biosynthesis of monolignols. Genomic and reverse genetic approaches. Phytochem Rev 2 289-306... 

The reverse genetic approach relies on the construction of suitable libraries. The initial step is to homogenize the tissue and isolate the mRNA population. As mRNAs are single stranded and relatively unstable, they are first converted to their complimentary DNA (cDNA) sequence, and then into the more stable double-stranded cDNA molecule. The next step is to find a suitable vector that can carry the cDNA molecule and replicate itself multiple times. This will allow each message to be amplified to the large numbers of copies required for isolation and purification. 

Having demonstrated that there are multiple Arabidopsis GBF proteins, a difficult task now will be to determine the function of these individual proteins. In order to address this point directly, clearly a genetic, or reverse genetic approach will be necessary. 

Several model plants, including 0. sativa, A. thaliana, Z. mays, G. max, and Medicago truncatula, for which genome information is currently available,219 are a rich source of antimicrobial metabolites. Genetic and reverse genetic approaches are providing evidence for the biological importance of antimicrobial compounds in host defense mechanisms. 

After their introduction, the transposable elements are allowed to propagate resulting in several collections of plants containing multiple transposon inserts at different locations in the plant genome. Apart from being a source of mutants for specific screens, these collections are now a valuable source for reverse genetics approaches (see below). 

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