Revision as of 02:28, 1 November 2017

• 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 mKate FF4 Reporter

The first reporter we tested is what we call the mKate FF4 reporter.

The mKate FF4 reporter features the red fluorescent mKate gene split into two exons, and has an intron that contains 3 microRNA sites for FF4 in between these two exons. We used this split mKate 2-exon construct as a reporter construct that we used to determine if our dCas13 or Ms2 systems were successful in hindering the splicing of the FF4 intron. Upstream of these sequences is a either the constitutive hEF1a promoter or the DOX controlled TRE-tight promoter.

Lucky for us, the entry vector version of this reporter were available to us via the Weiss Lab. After inserting the hEF1a and TRE-tight promoters, the reporter could be used in transfections.

We used this reporter to

In the absence of dCas13a or Ms2, the intron should be spliced out as normal, leading to a complete mKate mRNA transcript and flouresence would be seen.

In the presence of dCas13a or Ms2, which targets the 3' splice site of the intron, the second exon would be spliced out along with the intron, and the final mRNA transcript would not include the second exon. Thus, with out system, we expect a knock down of flourescence.

Results

ASO and MS2 Results

Before testing the tiling of our ASO guides, we needed to determine the ideal amount of mKate, Ms2, and ASO required to see the effects of our sytem.

mKate Titration

As discussed above, ASOs are unstable in the cell. Therefore, we needed to ensure the proper ASO to reporter ratio. We suspected that our system would be able to affect the splicing of mKate up to a threshold, after which the mKate would saturate the system. These expected results can be seen below. For a detailed explanation of how to read a CytoFlow graph click here

In our experiment, we varied the amounts of mKate-ff4 from 10 to 500 ng using ASO3 and ASO2+. The ASO2+ was co-transfected with Ms2. For a detailed explanation of how to plan a mammalian transfection click here

Caption: mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bin of each result.

For the mkate titration for ASO3, we observe a standard mkate curve for all reporter concentration above 100 ng. Below 100 ng there was a disturbance in the normal curve, indicating that our system might be acting on the system.

For the mkate titration for ASO2+, we also observe a disturbance at 100 ng of reporter.

Based on these results, we decided to transfect with 100ng of mkate-ff4 moving forward.