CRISPR-Edited Cell Lines to Identify Specific Transcripts for Per1 in Learning and Circadian Rhythms
Open Access
- Author:
- Bernhardt, Alicia
- Area of Honors:
- Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Janine Kwapis, Thesis Supervisor
Stephen Wade Schaeffer, Thesis Honors Advisor - Keywords:
- Per1
Circadian Clock
Memory - Abstract:
- Long-term memory is known to require de novo transcription. Previous research has determined that the gene Period 1 (Per1), which plays a well-established role in the circadian rhythm, is also needed for long-term memory within multiple memory-relevant brain regions such as the hippocampus. How Per1 modulates memory, however, is unclear. To address this question, here we aimed to create CRISPR-edited cell lines that have edited promoter regions to allow for the identification of specific transcripts of Per1. I investigated: (1) if the binding of the transcription factor Creb to CRE sites in the Per1 promoter region leads to the expression of a learning-specific Per1 transcript; and (2) if the binding of the heterodimer Clock/Bmal to E-boxes in the Per1 promoter region leads to the expression of a circadian rhythm-specific Per1 transcript. To meet this objective, I first designed guide RNAs (sgRNAs) to allow for the editing of the 5.5 kb promoter region. Second, the cutting efficiency of sgRNAs was determined by transfecting a mouse hippocampal cell line (HT22). Third, donor plasmids will be designed and cloned to contain either mutated E-boxes or CRE sites. Fourth, the mouse hippocampal cell line will be transfected with the sgRNAs, Cas9, and the appropriate donor plasmid to create the edit. Fifth, the DNA will then be extracted to analyze the promoter region for the presence of mutations using Sanger sequencing. I will be looking for a homozygous mutant cell line, but as this is a large edit and unlikely to occur in a single editing process, the process will be repeated until a homozygous mutant cell line is achieved. Future work will create two mice lines that will contain the mutant promoter regions specifically in the hippocampus and then their learning behaviors will be evaluated, tested, and analyzed in addition to assessing their circadian rhythm.