revolutionized gene analysis by allowing genes to be expressed from the same recombina-
tion cassette inE. coli, Saccharomyces cerevisiae, or baculovirus expression systems (Liu
et al. 1998) (Invitrogen Carlsbad), providing easier access to tools that broaden the scope for
the functional analysis of genes.
7.4.4 Vectors for Promoter Analysis
Tools that identify the spatial and temporal expression patterns of genes also provide
important clues in functional genomics studies. Frequently, vectors are designed to allow
the promoter orciselement of a GOI to be fused upstream of a reporter-coding sequence
(reporter genes are discussed in Chapter 9). Such constructs are used to determine the
cell type(s), organ type(s), or developmental stage in which a gene is expressed. By assem-
bling promoter ENTRY clones in recombination-compatible vectors, researchers are com-
piling a library of promoters and enhancers that are universally compatible with a wide
variety of vectors. The modular assembly of DNA components has recently been extended
through the introduction of additional novel recombination sites (Multisitew,Gatewayw,
Invitrogen) with unique specificities that allow multiple DNA fragments to be assembled
in a single vector (Fig. 7.19). This facilitates the simultaneous incorporation of a promoter,
ORF, and epitope tag into a single plant vector derived from collections of the modular
component parts.
Figure 7.18.Gene silencing in plants can be achieved using inverted repeat transgene constructs that
encode a hairpin RNA (hpRNA). Using GatewayTMcloning technology, the production of such
inverted-repeat transgene constructs can be achieved efficiently, since DNA fragment orientation
during the excision and integration process is maintained and the GatewayTMrecombination cassettes
are arranged in opposite orientations with respect to each other.
180 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION