I have spent the past few weeks trouble shooting the NF1 guide RNA cloning process. My first few attempts resulted in zero viable colonies, however, I was able to successfully obtain colonies containing constructs from 7 of 8 guide RNAs. These plasmids were sent for sequencing and each contains the proper insert. I will begin testing the efficacy of these guide RNAs in HEK293T cells soon. One of the foreseeable problems is that NF1 knockouts will be difficult to verify since the current selection of effective antibodies against NF1 is very poor. I will be testing a panel of antibodies since we have receive NF1 wild type Schwann cells to use as positive controls. NF1 null MPNST lines will serve as negative controls. In the case that an effective antibody is not found, we will resort to quantitative reverse transcriptase PCR and sequencing to verify knockouts.
I have also begun testing NF1 wild type and NF1 heterozygous cell lines for SIRT5 dependency. Preliminary results suggest that NF1 wild type cell lines are less sensitive to SIRT5 depletion than NF1 mutant cell lines. In addition to benchwork, my PI has suggested that we write a literature review on MPNST. I have written a first draft in the past few weeks and I will soon begin the editing process. Thus far, the Summer has been more productive than I could ever have imagined. I look forward to seeing what I can accomplish in the next 8 weeks.