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HC70AL Oral Presentation. Shirin Oloumi Spring 2004. Arabidopsis thaliana Knockout Project. AT3G12480 Located on the 3 chromosome Forward orientation 6 exons, 2 UTRs 2636 base pairs long 3001 base pairs long when amplified with forward and reverse gene specific primers.
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HC70ALOralPresentation Shirin Oloumi Spring 2004
Arabidopsis thaliana Knockout Project • AT3G12480 • Located on the 3 chromosome • Forward orientation • 6 exons, 2 UTRs • 2636 base pairs long • 3001 base pairs long when amplified with forward and reverse gene specific primers. • Forward and reverse primers roughly 400 base pairs before/after my gene, each 29 bps long • Transcription factor, putative • DNA binding protein • Expressed protein is 293 amino acids 3001 base pairs long 5’ 3’ 1 2636 -436 2987
Location in Arabidopsis Chromosomal coordinates: 3958744-3960479 Overlapping 610 bps 1009 bps 3’ 5’ 3’ 5’ AT3g12470 AT3G12480 AT3G12490 5’ 3’
Gene Activity in SRB and Arabidopsis RT-PCR Gel • My gene is active in leaf, stem, ovule, 14 day embryo, 30 day axis, and flower, although that band is very faint. control cDNA • Arabidopsis transcription levels • Activity of gene declines as plant grows • High activity in Post Maturation Green Seeds,
Will knocking out my gene affect plant’s development in any way? Do I have a TDNA knockout in my gene? Screening Madison Lines FW with Superpools Reamplification of Superpool 5 and DNA pools 6, 7, 8 confirms that DNA pool 7 contains the knocked out form of my gene. RV with Superpools Superpool 5 DNA Pool #7 FW and RV Blot • Superpool 5 amplified with the forward primer contained a knockout, not visible in the gel, but obvious in the autoradiogram. Superpool 11 with reverse primer did not contain a knockout worth pursuing. There was also a hit Reamplification of Superpool 5 shows that its contains a fragment 400 bps long, which was the fragment I expected. Reamp of Superpool 5 and control TDNA This shows the rough approximation of my TDNA insertion site in my gene for Superpool 5. 3’ 5’ control Superpool 5 -36
Will knocking out my gene affect plant’s development in any way? Do I have a homozygous TDNA plant? Screening SALK Line 070404 051145 070404 • SALK Facility: • Line 051145: Insertion Site at +65 in 5’ UTR • Line 070404: Insertion Site at +299 in Intron 1 5’ 3’ TDNA Allele in Plants 1-9 + WT (right side) Wild Type Allele in Plants 1-9 + WT (right side) The first gel amplified the wild type allele, if any. Every one of my plants contained a wild type allele. However, the results were not conclusive enough, so I repeated the experiment with two reactions, one PCR with tubulin primers and one without. The gel turned out to be very clear without tubulin. This gel amplified my gene if it contained a TDNA insert. I did not get anything other than tubulin, which suggests that my reaction worked. Upon repeating the experiment, the data showed that I indeed did have heterozygous plants. Unfortunately, no homozygous TDNA showed up.
Will knocking out my gene affect plant’s development in any way? Do I have a homozygous TDNA plant? Screening SALK Line 070404 Data was inconclusive from previous experiment with plants 1-9. Round two results:
The next step… Wild Type Arabidopsis flower. Mutant Arabidopsis flower. • Continue looking for homozygous TDNA in SALK lines. Observe heterozygous phenotypes for any abnormalities. • Narrow down Madison lines to 25 Seed Pools and then on to one line/plant. Observe phenotypes of these plants.
