no longer a practical approach to facilitate the analysis of all the genes and promoters
from these model organisms. Plant vectors compatible with Gateway recombination
cloning and Univector recombination cloning have been generated to aid these analyses
(Fig. 7.17).
Recombination-compatible collections of plant ORFs (open reading frames; a sequence
encoding a polypeptide) have also been generated. Trimmed ORFs lacking 5^0 or 3^0 UTRs
(i.e., containing protein-coding sequences only) can be shuttled rapidly and efficiently
between vectors bearing compatible recombination sites. These so-called ORFeome collec-
tions have been generated so that the positions of the original translation initiation and
termination codons remain intact (“closed” ORF configuration). However, since some
applications to investigate gene function require the addition of C-terminal peptide
fusions, ORFeome collections in which the stop codon is omitted (“open” ORF configur-
ation) are also being generated. Often, the initial functional data on an ORF or gene are on
the phenotype it induces when it isectopicallyexpressed (i.e., in tissues in which it is not
normally expressed) under a constitutive and near-ubiquitous promoter. Gateway vectors
designed for this type of analysis have been generated using the strongly active 35Spromo-
ter from cauliflower mosaic virus (CaMV). Some of these vectors have an additional design
feature that provides stop codons adjacent to the 3^0 recombination site in all three reading
frames, to facilitate the expression of open as well as closed ORF configurations. Of course,
not all ORFs can be misexpressed constitutively. Some cause lethal effects when expressed
in this manner. In such cases, ORFs can be shuttled into vectors that are designed for con-
ditional or inducible ectopic expression (Karimi et al. 2002; Curtis and Grossniklaus 2003;
Joubes et al. 2004) or even to vector systems that allow induced expression in restricted cell
types (Brand et al. 2006).
7.4.2 Vectors for RNA Interference (RNAi)
A very powerful tool that helps elucidate gene function is to reduce, or “knockdown” native
gene expression in the organism using RNA interference (RNAi) (Waterhouse et al. 1998).
Here, double-stranded RNA is produced by the transcription of an inverted repeated
sequence of a gene. This transcript forms a hairpin–loop structure that triggers the RNAi
pathway, leading to the degradation of homologous mRNAs [reviewed by Brodersen and
Voinnet (2006)]. The careful construction of specialized GatewayTMdestination vectors
guarantees the rapid and efficient production of double-stranded RNAs (Fig. 7.18). In stan-
dard GatewayTMvectors, theattsite modifications were designed to maintain DNA frag-
ment orientation during the excision and integration process (Hartley et al. 2000). The
arrangement ofattsites in RNAi constructs ensures the easy insertion of two identical
gene segments in opposite orientations, downstream of a constitutively active promoter
(Fig. 7.18). Constitutively expressed interfering RNA can be used to silence genes through-
out a plant’s development, or can be expressed conditionally to provide temporal control
over the onset of gene silencing.
7.4.3 Expression Vectors
The thorough analysis of gene function frequently involves expressing a GOI, not only in
plants but also in multiple systems. With traditional cloning methods, independently
derived expression constructs must be made. Recombination cloning technology has
7.4. VECTOR DESIGN 179