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

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”

The Reverse Genetics Approach

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?

How Does Our Gene Affect Embryo Development? Genotyping Plants Why genotype? What does it show us? Approach: 1)Grow SALK/SAIL seeds 2)Isolate genomic DNA 3)Use PCR to determine genotype 4)Sequence bands to find T-DNA insertion site

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

How Does Our Gene Affect Embryo Development? Phenotyping Plants How can we find a mutant phenotype? What are we looking for? Approach: 1)Observe plants in greenhouse 2)Observe siliques/seeds with microscope 3)Observe embryos using Nomarski Microscope

How and Where is Our Gene Expressed? 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?

RT-PCR 1.Isolate RNA from plant 2.Use Reverse Transcriptase to synthesize cDNA 3.Use gene-specific RT-PCR primers to amplify DNA 4.Visualize brightness with gel electrophoresis

Promoter Study Used to pinpoint gene expression to exact areas of plant 1.Amplify promoter region 2.Put in vector 3.Transform E. coli 4.Grow and select for colonies 5.Verify sequence of promoter region 6.Further study with GFP

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