1. HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana
Genes
AT4g27410
AT5g07680
Salk Lines # #
Presented By Tim Schulz

2. -----------------------
Outline
Overall: What will knocking out these genes tell us about how they function?
What are the characteristics of the genes and T-DNA inserts?
Plant Genotypes?
Plant Phenotypes?
What Next?

3. Structure & Location of AT4g27410
 .5kb (Salk)
(bp)
Promoter
UTR
Exon
Intron
Exon
UTR
Flanking Sequence 3’ 5’
+ 167
FW 
- 331
 RV
~1.4kb Between Primers (w/o T-DNA)
~ 1.6kb Entire Gene (w/o T-DNA)

4. Structure & Location of AT5g07680
 .5kb (Salk)
(bp)
UTR
Exon
Intron
Exon
Intron
Exon
UTR
Flanking
Sequence 3’ 5’
FW 
 RV
~750bp (w/o T-DNA)
~ 1.6kb Entire Gene (w/o T-DNA)

5. Actual T-DNA Locations
AT4G27410 – Different From SALK
AT5G07680 – Same as SALK
Salk’s Report for AT4G27410:
 .5kb (Salk)
(bp)
Promoter
UTR
Exon
Intron
Exon
UTR
Flanking Sequence

6. Actual T-DNA Locations
AT4G27410 – Different From SALK
AT5G07680 – Same as SALK
My Results for AT4G27410 Show:
 .5kb (Salk)
 .5kb (Salk)
(bp)
Promoter
UTR
Exon
Intron
Exon
UTR
Flanking Sequence

7. Actual T-DNA Location: AT4G27410 PCR
Odd Results From FW + RV + LBb1:
Plants 6-13

8. Solution: Separate Primers
LB+FW, LB+RV, RV+FW

9. Actual T-DNA Location: AT4G27410
Conclusion After - Band-Size Analysis - Sequencing:
 .5kb (Salk)
 .5kb (Salk)
(bp)
Promoter
UTR
Exon
Intron
Exon
UTR
Flanking Sequence

10. Genotypes: First SALK Line
AT4G27410
Plant
1. W/T 2. Heterozygous 3. W/T W/T 5. W/T 6. Homozygous 7. W/T 8. W/T 9. Heterozygous 10. W/T 11. W/T 12. Heterozygous 13. Homozygous 1 2
1. W/T
3. W/T
5. W/T
7. HOMO
2. HET
4. W/T
6. HOMO
8. W/T 3 4
9. HET
12. HET
11. W/T
10. W/T
13. HOMO

11. Genotypes: Second SALK Line
AT5G07680
Questions:
Missing Plants?
Smaller Plants?

12. Phenotypes? Smaller Plants – NAM Related?
Missing Plants – Embryo Lethal?

13. Genotypes: Second SALK Line
Plants #

14. Smaller Plants: Mutants?

15. Smaller Plants: Mutants?

16. Smaller Plants: Mutants? The Test: #10 & #24
(Hetero DNA for Positive Control)
~750bp
 T-DNA

17. Genotypes: Second SALK Line
AT5G07680 1 2
Plant
1. W/T 2. W/T 3. W/T W/T 5. Heterozygous 6. W/T Heterozygous 9. Heterozygous 10. W/T 11. W/T 12. W/T 13. Heterozygous 24. W/T
1. W/T
3. W/T
5. HET
7. (Too Small)
2. W/T
4. W/T
6. W/T
8. HET 3 4
10. W/T
12. W/T
9. HET
13. HET
11. W/T

18. Phenotypes? Smaller Plants – NAM Related?
Missing Plants – Embryo Lethal?

19. Missing Plants – Embryo Lethal? Analysis through Microscopy
Heterozygous
Silique Image 1
All Alive: No Immediate affect
3:1 Alive/Dead Ratio- Seed Development affected Zygotically
1:1 Alive/Dead Ratio- Female Gametophyte affected, post fertilization

20. Missing Plants – Embryo Lethal? Analysis through Microscopy
Heterozygous
Silique Image 2

21. Missing Plants – Embryo Lethal? Analysis through Microscopy
Heterozygous
Silique Image 2
(opened)

22. Missing Plants – Embryo Lethal? Heterozygous-Embryo Development
Early Heart

23. Missing Plants – Embryo Lethal? Heterozygous-Embryo Development
Heart

24. Missing Plants – Embryo Lethal? Heterozygous-Embryo Development
Late Heart

25. Missing Plants – Embryo Lethal? Heterozygous-Embryo Development
Torpedo

26. Missing Plants – Embryo Lethal? Heterozygous-Embryo Development
Walking
Stick

27. Missing Plants – Embryo Lethal? Heterozygous-Embryo Development
U-Turn

28. Where to go from here… Promoter + GFP
Multiple Knockouts (determined via microarrays)
Other, non-knockout related experiments

29. -----------------------
Conclusion
Primary Goal  Know Gene Functionality
Primary Method  T-DNA Knockout
What are the characteristics of the genes and T-DNA inserts?
Plant Genotypes?
Plant Phenotypes?
What Next?