hairpinRNAi vectors for plants
CSIRO hairpinRNAi vectors are designed to simplify the use of hairpinRNAi in functional genomics and trait development.
Academic, not-for-profit organisations may obtain these vectors free of charge for research use. Material Transfer Agreements can be downloaded from www.pi.csiro.au/tech_licensing_biol/GeneSilencingVectors.htm
CSIRO vectors:
Cloning by restriction sites
| Dicots |
High throughput cloning through Gateway™ recombination (Invitrogen)
| Dicots |
|
Novel promoters |
pHANNIBAL/pKANNIBAL
pHANNIBAL (with bacterial ampicillin resistance) and pKANNIBAL (with bacterial kanamycin resistance) are designed for directional insertion of PCR products on either side of the PDK intron.
A fragment from the target gene is amplified in a PCR reaction, appended with the restriction sites and cloned sequentially on either side of the intron to become the two arms of the hairpin. The ideal target sequence is at least 300 nucleotides in length.
The hairpin cassette is then cloned into a convenient binary vector in a single step using the NotI restriction sites.
The pKANNIBAL vector is particularly useful in cloning fragments maintained in ampicillin resistance vectors without additional purification steps.
These vectors are preferred for silencing small numbers of target genes.
Figure 1. hairpinRNAi vector for dicots
Figure 2. Construction of hairpinRNAi vectors using pHANNIBAL
pSTARLING
This is a pUC based vector with a novel set of restriction sites. The NotI fragment needs to be cloned into a binary vector.
This vector uses the maize ubiquitin promoter for high level constitutive hairpinRNAi production in monocot plants. If Agrobacterium transformation is used, the NotI fragment must be cloned into a suitable binary vector.
Figure 3.
pHELLSGATE
pHELLSGATE vectors use Gateway™ recombinational cloning (Invitrogen) for high-throughput construction of hairpins targeting hundreds to thousands of genes. The pHELLSGATE vectors are particularly useful for functional genomics applications, such as determining the functions of individual members of a gene family, or genes involved in complex biochemical or developmental pathways, or even whole genomes.
Gateway™ provides directional cloning of a PCR product targeting each gene along with a negative selection marker (ccdB) that ensures both halves of the hairpin are present in the construct.
PCR products for each gene are generated with flanking attB1 and attB2 sites which are recombined into a plasmid with attP1 and attP2 sites. A second recombination reaction inserts the target sequence into attR1 and attR2 sites in the pHELLSGATE vector. By using a two step cloning procedure, small attB sites are generated in the final construct. The single step recombination reaction generates longer attL sites and silencing efficiency is significantly reduced.
pHELLSGATE 12 contains two introns in opposite orientations so that the final product of recombination always contains one spliceable intron regardless of the orientation of the Gateway™ recombination reaction.
The PCR, recombination, and transformation steps to produce these vectors can be performed in a 96-well format, making them suitable for the automation required for large scale projects.
Download
pHELLSGATE sequence (sequence
is a Vector Nti file)
Click link to view; right-click link and select
"Save Target As..." to save file
Figure 4.
Figure 5.
Click to enlarge
Figure 6. pHELLSGATE 12 is designed with dual introns such that all the clones contain an intron in the proper orientation
pSTARGATE
This vector is a modification of
pHELLSGATE
for use in monocot plants
and uses the same Gateway™ recombination system. It contains an
ubiquitin promoter and its intron along with a hygromycin resistance gene.
Download predicted sequence of pSTARGATE (sequence is a pDRAW file) This program can be downloaded for free from www.acaclone.com. Note, pSTARGATE has not been sequenced.
Click link to view; right-click link and select "Save Target As..." to save file
Figure 7.
pWATERGATE
In this vector the 35S promoter in pHELLSGATE is replaced with the Arabidopsis RbcS promoter.
Many Arabidopsis insertion mutant libraries (e.g. Salk, FlagdB) have been generated using T-DNAs incorporating the 35S promoter. Super-transforming with plasmids encoding 35S-driven hairpinRNA runs the risk of producing weak RNAi due to cross silencing of the 35S promoter. pWATERGATE has been produced to overcome the problem by regulating hairpinRNA with the highly active ARbcS promoter.
This feature removes the possibility promoter silencing when super-transforming transgenic plants generated using traditional promoters.
Download pWATERGATE sequence (sequence is a Vector Nti file)
Click link to view; right-click link and select "Save Target As..." to save file
Figure 8.
pOpOff
Figure 9. |
CSIRO has developed a vector system that gives dexamethasone-inducible RNAi against plant genes. The system utilizes a modified pHELLSGATE vector, under the control of the pOp6 promoter (developed by Ian Moore, University of Oxford) and the synthetic transcription factor, LhGR.
The production of hairpinRNAi from this system can be turned on and off by the application and removal of dexamethasone.
The inducibility of hairpinRNAi from this system may be very useful in helping to identify the functions of genes which when constitutively silenced give embryo lethality or complex phenotypes.
Download pOpOff
sequence (file
is readable in Microsoft Word)
Click link to view; right-click link and select
"Save Target As..." to save file
Figure 10. Maps
of pOpOff vectors with suggested sites for diagnostic restriction
digests following recombination of target gene fragments to replace
the ccdB gene. The vector backbones (not shown) are >10kb in
size. Restriction fragment sizes are approximate and given in kb.
Plant Biotechnology Journal (2005)
3: 583-590.
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