Team:MIT/REST-results

• ABOUT US
  Team Attributions
• BACKGROUND
  Alternative Splicing CRISPR RNA Binding Proteins REST
• PROJECT
  Guides and ASOs mKate-FF4 2 Exon mKate HBG 3 Exon mKate HBG 3 Exon Dual Fluorescence
• LAB WORK
  Parts/Improved Parts Protocols Notebook Future Work
• MODELLING
• HUMAN PRACTICES
  Integrated HP Public Engagement Collaborations InterLab
• AWARDS
  HP Silver HP Gold Education & Public Engagement Model Parts Collection

Experiments with Small Cell Lung Cancer

Once we had decided on a disease model, namely Small Cell Lung Cancer, we needed an in-vivo way of testing our system. To this end we obtained the H82 Cell line from ATCC.

Preliminary Results

Viafect Optimization

The first thing we had to ensure was that we were able to get DNA into the new cell line. We performed a Viafect optimization to test what our ratio of plasmid to transfection reagent should be. We transfected constitutively expressed BFP and reagent at ratios 1:1, 1:2, 1:3 and 1:4.

Viafect Optimization

Ratio of Viafect:BFP vs. the blue fluorescent output (AU) of the H82s

We see the most fluorescent output at a 1:3 ratio of Viafect:BFP. Moving forward, we transfected at a 1:3 ratio.

RT-qPCR

To analyze the effectiveness of our RNA binding protein system on the alteration of the splicing event of REST in H82 Small Cell Lung Cancer cells, we conducted to Real-Time PCR on H82 SCLC cell line samples, transfected with the dCas113a along with the constructed REST1, REST2, REST3, REST4, REST5 and HBG (random) Guides respectively. The RT-qPCR experiments allow us to assess the amounts of each RNA isoform present in the H82 cells. We have carried out three selective amplifications for each of the 6 samples above:

1. First Round of Amplification is selective Amplification for REST using primers spanning the alternative exon junction but not the exon itself.
2. Second Round of amplification for REST4 is by using primers spanning the alternative exon.
3. Third round of amplification is the selective amplification of Beta Actin gene as a housekeeping gene

We can see from these results that REST Guide 2 and REST Guide 4 performed the best.