Modulating the body's networks could become mainstream therapy for many health issues.
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Edited by Nina Fedoroff, Pennsylvania State University, University Park, PA, and approved June 10, 2005 (received for review March 30, 2005)
Article Figures & SI
Figures
- Fig. 1.
Constructs used in gene-targeting experiments in Arabidopsis. (a) Construct pHS-35SRAD54 contains the ORF of the S. cerevisiae RAD54 gene (ScRAD54-ORF) under the control of the cauliflower mosaic virus 35S promoter (35S PRO) and the transcription termination signals of the Octopine synthase gene (OCS-3′). LB, left border; RB, right border. (b) Construct pHS-GT1, the gene-targeting vector, contains the Cru box corresponding to the 1,210-bp genomic sequence in the 5′ end of the Cruciferin gene. The Cru box lacks the ATG initiation codon and 36 bp downstream of the ATG. Upstream of the Cru box is the glufosinate (BASTA) resistance gene transcribed in opposite orientation to the Cruciferin gene (arrows). The GFP reporter is fused in-frame downstream to the Cru box and is followed by the transcription termination sequence of the Nopaline synthase gene (NOS). A 2,492-bp fragment identical to the genomic region downstream of the Cru box flanks the GFP reporter. This fragment contains 773 bp corresponding to the 3′ end of the Cruciferin gene (ciferin) followed by 1,719 bp of downstream noncoding DNA. Constructs pHS-35SRAD54 and pHS-GT1 were cloned in the backbone of the T-DNA binary vector pMBLArt in between the left and right borders of the vectors (LB and RB, respectively).
- Fig. 2.
The seed-based gene-targeting assay. The assay is based on the activation of GFP upon homologous integration of the gene-targeting vector (pHS-GT1, described in Fig. 1) into the genomic DNA (gDNA) of the Arabidopsis Cruciferin gene. Such events are identified by visualization of green fluorescent seeds under a fluorescent microscope (see black box with green seeds on the bottom right). The vector is a linear T-DNA sequence replacement vector with homology to the target Cruciferin gene on both sides of the vector (1,210 and 2,492 bp, respectively). The gene-targeting product gives rise to a chimeric Cru-GFP fusion protein expressed in the seed under the control of the Cruciferin promoter. NOS, Nopaline synthase.
- Fig. 3.
Validation of gene-targeting events by PCR. To confirm the precise integration of pHS-GT1 into the Arabidopsis genome, we used two primer pairs: one pair (black arrows) for the 5′ border and the other for the 3′ border (white arrows) on genomic DNA template from plants derived from green fluorescent seeds. In both cases, one primer anneals to the genomic DNA beyond the gene-targeting vector, and the other primer is homologous to the GFP sequence. (a) The 5′ PCR product yielded a 3,136-bp fragment for a sample of 3 of the 15 plants analyzed (the right lane is a molecular weight marker). (b) Amplification of the 3′ integration junction of the same three plants, yielded the expected 3,397-bp fragment. The PCR fragments were sequenced, indicating precise integration from both sides of the vector.
- Fig. 4.
Validation of gene-targeting events by Southern blot analysis. A Southern blot analysis was performed by using the Cru box as a probe (black rectangle) on genomic DNA from plants derived from green fluorescent seeds (GT) and WT seeds (WT) digested by HindIII. This analysis was done to confirm homologous integration of pHS-GT1 into the Arabidopsis genome from both 5′ and 3′ ends and to estimate the copy number of pHS-GT1 insertions into the genome. (a and b) The structure of the WT allele (a) and the targeted allele (b) is shown. (c) As expected for precise gene-targeting events, fluorescent seeds were heterozygous, showing one band (2,236 bp) corresponding to the gene-targeting allele (lane GT) and another band (6,067 bp) to the WT allele. DNA from WT seeds (lane WT) showed only the 6,067-bp band. These data suggest that plants shown in the GT lanes are heterozygote and contain a single copy of the vector at the CRUCIFERIN locus. (d) Germinal transmission of the targeted allele was tested by Southern blot analysis in the progeny of these heterozygous plants. The Cru probe was hybridized to HindIII-digested genomic DNA from self-pollinated progeny of the targeted plants shown in c. A sample of 16 plants of the 27 tested is shown. Of the 16 lanes shown, the first 12 (from the left) are from one gel and the last four are from another gel. Five of the 27 tested plants showed a single band of ≈2,236 bp, as expected for the homozygote-targeted allele (Hom lanes). The remaining 22 plants showed two bands as expected for heterozygotes (Het lanes).
Tables
- Table 1. Summary of gene-targeting experiments with the seed assay in RAD54-expressing and WT plants
No. of seeds Exp. Plant type Total scored Transformed Fluorescent (putative targeting events) Gene-targeting frequency (fluorescent/transformed) Fold gene-targeting enhancement (RAD54/WT) I WT 300,000 4,500 29 6.4 × 10−3 5 RAD54 415,000 6,225 197 3.1 × 10−2 II WT 95,000 1,425 8 5.6 × 10−3 25 RAD54 42,000 630 88 1.4 × 10−1 III WT 225,000 4,500 4 8.8 × 10−4 62 RAD54 132,000 2,640 146 5.5 × 10−2 IV WT 35,000 735 7 9.5 × 10−3 18 RAD54 21,500 451 77 1.7 × 10−1
High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene
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