SUPER RESOLUTION MICROSCOPY TO INVESTIGATE INTRACELLULAR LOCALIZATION AND INTERACTION FOCI OF PROTEINS OF INTEREST
Interaction studies of two proteins labeled with Atto488 & Alexa647 by conventional TIRF and super resolution STORM microscopy in a mammalian cell (A) Principle of N-STochastic Optical Reconstruction Microscopy [STORM](adopted from nikon webpage) (B) Total Internal Reflection(TIRF) image of the double labeled (Atto488&Alexa647) HEK-293cells.(C) Reconstructed two color STORM (super-resolution) image from: a data set of 20,000 frames visualized by N-STORM microscope.(D) Higher magnification views of the closely spaced two different protein molecules. Each localization is depicted in the STORM image as a Gaussian peak, the width of which is determined by the number of photons detected. (E) Average distance between two proteins molecules (spatial distribution) calculated by Matlab program.(F) N-STORM Super Resolution Microscope. Scale bars-10 μm (B), 100nm(C) and 50 nm (D).
TRANSCRIPTOME RECONSTITUTION OF RNA EXOSOME MUTANT PRIMARY CELLS USING RNA-SEQUENCING AND BIOINFORMATICS PIPELINES
Transcriptpme assembly of Exosc3 and Exosc10 ablated B cells and ES cells. Stepwise depiction of bioinformatics pipeline and parameters utilized for analyzi the transcriptomes of Exosc3COIN/COIN ROSA26CreERt2/+ or Exosc10COIN/LacZ ROSA26CreERt2/+ B cells and ES cells.
Conditional-Inversion (COIN) allele mediated genome engineering of mouse model systems
Exosc3 deficient Bcells are defective in immunoglobulin diversification (A) Schematic of the Exosc3COIN allele and conversion to Exosc3COINinv. Cre mediated inversion of the Lox2372 pair (red triangles) and subsequent deletion via the LoxP pair (violet triangles). GFP expressing terminal exon represented by green arrows. Exons are represented as numbered boxes. Splicing pattern indicated by dashed lines. SA, splice acceptor. (B) Southern blot of HindIII digested genomic DNA from naive splenic B cells (day 0) or 4-hydroxytamoxifen (4-OHT) treated (days 2-4), LPS + IL-4 stimulated B cells from Exosc3COIN/+ and Exosc3COIN/COIN mice on ROSA26CreERt2/+ background. Radiolabeled probe is specific for exon 3 of Exosc3. (C) Quantitative RT-PCR time course analysis of Exosc3 mRNA expression in naive (day 0) or 4-OHT treated (days 2-4), LPS + IL-4 stimulated B cell cultures. Indicated Exosc3 genotypes are on ROSA26CreERt2/+ background. Expression levels are normalized to cyclophilin (Ppia) and plotted relative to naive Exosc3COIN/+.