Dr. Jennifer Wilking 2010 Recipient Research Update

December 17, 2010
Progress report for Jennifer A. Wilking

Dr. Jennifer Wilking

Dr. Jennifer Wilking

MIND foundation fellowship Proposal: Examine nicotine induced epigenetic changes that lead to gene expression differences
Brief description of progress in the past few months:

  • Wrote and submitted the necessary animal protocols.
  • Determining the most effective technique for extracting high quantity and quality of DNA and RNA from the same brain samples.
    • Trying several different methods and optimizing those methods
  • This will allow for a more direct analysis of nicotine induced epigenome and transcriptome changes.
  • Additionally, obtaining high quantity and quality DNA/RNA will not only improve the analysis but could possibly prevent a whole genome wide amplification which might bias the data.
  • Optimized DNA sonication (shearing of DNA into specific size) settings
  • Sonication settings are particularly important to for examining methylation differences. The size of DNA will directly affect the efficiency of DNA methylation assays.
  • Several recent papers were published which evaluated methods for exploring differential DNA methylation
  • Based on these new findings, we have re-evaluated the chosen method for examining DNA methylation changes
  • MBD (methyl binding domain assay) vs MeDIP (methyl-DNA immunoprecipitation assay)
  • Findings suggested that using MBD assays achieve higher overall enrichment compared to MeDIP, especially in CpG-rich regions
  • As a result, we will examine differential DNA methylation changes using a MBD assay
  • I am in the process of testing different MBD assays
  • Following isolation of the differentially methylation DNA, we will use a microarray to interrogate differences in methylation patterns following nicotine exposure.
  • As a result, we are working with a company to minimize the amount of starting material (DNA) required
  • This company has offered us several microarrays to test the minimum amount of DNA required for reproducible results using their arrays
  • As a result, we are optimizing DNA isolation, sonication, and DNA methylation recovery in order to test the ideal conditions to be used on with arrays
  • Data mining GeneNetwork (an online web-based tool to analyze systems genetics) to determine potential baseline differential gene expression patterns between the two proposed inbred mouse strains.
  • This will allow me to create lists of genes that naturally differ between the two proposed mouse strains
  • From GeneNetwork and with other online sources, I can predict which genes might be influenced by nicotine exposure as well as select candidate genes that might be differentially methylated.

Additionally, we have added a behavioural component to this study. Not only is it important to assess the molecular changes induced by nicotine, but ultimately we need to understand the effect that molecular changes on behavioural outcomes. As a result, several behaviours will be examined.

  • As we would like to assess several behaviours, I have been designing and optimizing a behavioural test battery paradigm.
  • These chosen behaviours are to assess a combination of behaviours including anxiety-like behaviours, working memory and exploratory behaviours.
  • More specifically we will test open field activity, novel object recognition, elevated plus maze, and passive avoidance.
  • These behaviours are likely to be affected by nicotine exposure.

Other:
I recently attended the National Institute for Drug Abuse (NIDA) meeting as well as the Society for Neuroscience meeting in San Diego. The NIDA meeting was largely focused on recent advances in the nicotinic field and as a result, was highly relevant to this project. Additionally, the SFN meeting was a wonderful opportunity to further discuss the most recent findings in the nicotinic field but also to attend sessions on epigenomics, addiction and mental health. Moreover, attending this meeting allowed me to meet and discuss this project with other investigators.

December 17, 2010
Progress report for Jennifer A. Wilking
MIND foundation fellowship Proposal: Examine nicotine induced epigenetic changes that lead to gene expression differences
Brief description of progress in the past few months:
? Wrote and submitted the necessary animal protocols.
? Determining the most effective technique for extracting high quantity and quality of DNA and RNA from the same brain samples.
o Trying several different methods and optimizing those methods
? This will allow for a more direct analysis of nicotine induced epigenome and transcriptome changes.
? Additionally, obtaining high quantity and quality DNA/RNA will not only improve the analysis but could possibly prevent a whole genome wide amplification which might bias the data.
? Optimized DNA sonication (shearing of DNA into specific size) settings
o Sonication settings are particularly important to for examining methylation differences. The size of DNA will directly affect the efficiency of DNA methylation assays.
? Several recent papers were published which evaluated methods for exploring differential DNA methylation
o Based on these new findings, we have re-evaluated the chosen method for examining DNA methylation changes
? MBD (methyl binding domain assay) vs MeDIP (methyl-DNA immunoprecipitation assay)
? Findings suggested that using MBD assays achieve higher overall enrichment compared to MeDIP, especially in CpG-rich regions
o As a result, we will examine differential DNA methylation changes using a MBD assay
? I am in the process of testing different MBD assays
? Following isolation of the differentially methylation DNA, we will use a microarray to interrogate differences in methylation patterns following nicotine exposure.
o As a result, we are working with a company to minimize the amount of starting material (DNA) required
? This company has offered us several microarrays to test the minimum amount of DNA required for reproducible results using their arrays
? As a result, we are optimizing DNA isolation, sonication, and DNA methylation recovery in order to test the ideal conditions to be used on with arrays
? Data mining GeneNetwork (an online web-based tool to analyze systems genetics) to determine potential baseline differential gene expression patterns between the two proposed inbred mouse strains.
o This will allow me to create lists of genes that naturally differ between the two proposed mouse strains
o From GeneNetwork and with other online sources, I can predict which genes might be influenced by nicotine exposure as well as select candidate genes that might be differentially methylated.
Additionally, we have added a behavioural component to this study. Not only is it important to assess the molecular changes induced by nicotine, but ultimately we need to understand the effect that molecular changes on behavioural outcomes. As a result, several behaviours will be examined.
? As we would like to assess several behaviours, I have been designing and optimizing a behavioural test battery paradigm.
o These chosen behaviours are to assess a combination of behaviours including anxiety-like behaviours, working memory and exploratory behaviours.
? More specifically we will test open field activity, novel object recognition, elevated plus maze, and passive avoidance.
? These behaviours are likely to be affected by nicotine exposure.
Other:
I recently attended the National Institute for Drug Abuse (NIDA) meeting as well as the Society for Neuroscience meeting in San Diego. The NIDA meeting was largely focused on recent advances in the nicotinic field and as a result, was highly relevant to this project. Additionally, the SFN meeting was a wonderful opportunity to further discuss the most recent findings in the nicotinic field but also to attend sessions on epigenomics, addiction and mental health. Moreover, attending this meeting allowed me to meet and discuss this project with other investigators.

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