What are the Methods and Approaches Used to Identify and Study Arabidopsis Seed Knock-Out Mutations?

1. What are the Methods and Approaches Used to Identify and Study Arabidopsis Seed Knock-Out Mutations? Eric Newton Garen Polatoglu Rena Schweizer

2. What are we trying to find out?* • What is the function of the gene? How does it affect embryo development? • How and where is the gene expressed? *Font color is “Arabidopsis Green”

3. The Reverse Genetics Approach

4. How Does Our Gene Affect Embryo Development? Growing knockout plants What is a knockout? Why do we use knockouts? Where do we get knockout seeds? How are the seeds made?

5. How Does Our Gene Affect Embryo Development? Genotyping Plants Why genotype? What does it show us? Approach: Grow SALK/SAIL seeds Isolate genomic DNA Use PCR to determine genotype Sequence bands to find T-DNA insertion site

6. Genotyping with PCR Use gene-specific forward and reverse primers, along with T-DNA primer

7. How Does Our Gene Affect Embryo Development? Phenotyping Plants How can we find a mutant phenotype? What are we looking for? Approach: Observe plants in greenhouse Observe siliques/seeds with microscope Observe embryos using Nomarski Microscope

8. Approach Reverse Transcription PCR (RT-PCR) Where in the plant is mRNA being produced? Promoter Study What does promoter do? How can it help us know expression of gene? How and Where is Our Gene Expressed?

9. RT-PCR • Isolate RNA from plant • Use Reverse Transcriptase to synthesize cDNA • Use gene-specific RT-PCR primers to amplify DNA • Visualize brightness with gel electrophoresis

10. Promoter Study Used to pinpoint gene expression to exact areas of plant Amplify promoter region Put in vector Transform E. coli Grow and select for colonies Verify sequence of promoter region Further study with GFP

11. Recap of HC70AL Procedures • Knock out gene • Identify knockout plants • Study phenotypic effects • Determine mRNA expression levels • Begin promoter study