Download Arabidopsis thaliana Knockout Project

HC70AL Oral Presentation
Shirin Oloumi
Spring 2004
Arabidopsis thaliana Knockout Project
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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
-436
2636
2987
Location in Arabidopsis
Chromosomal coordinates:
3958744-3960479
Overlapping
610 bps
1009 bps
5’
3’
AT3g12470
3’
5’
AT3G12480
AT3G12490
3’
5’
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
Quantative Transcription Levels of AT3G12480
•Arabidopsis transcription levels
1400
•Activity of gene declines as plant grows
1200
1000
Intensity
•High activity in Post Maturation Green
Seeds,
800
Wild Type
lec1
600
400
200
0
Stages
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
Reamp of Superpool 5 and
of Superpool 5
control
TDNA
shows that its
5’
control
contains a
fragment 400 bps
long, which was
Superpool 5
the fragment I
-36
expected.
This shows the rough approximation of my TDNA insertion site in
my gene for Superpool 5.
3’
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
5’
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.
SALK Facility:
• Line 051145: Insertion Site at +65 in 5’ UTR
• Line 070404: Insertion Site at +299 in Intron 1
3’
TDNA Allele in Plants 1-9 +
WT (right side)
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.