Journal: bioRxiv

Article Title: A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

doi: 10.1101/2021.08.17.456253

Figure Lengend Snippet: A , Overall proportion of different alternative splicing events in control and Ago2 cKO meiotic and post-meiotic germ cells. B , Change in exon percent spliced in (PSI) for individual transcripts with a significant difference in skipped exons in Ago2 cKO compared to control germ cells. C , Nuclear:cytoplasmic ratios in post-meiotic cells for two transcripts bound ( Ak7 , Hmgb2 ) and two transcripts not bound ( Ift27 , Ppib ) by nuclear AGO2. Localization was assessed by single-molecule RNA in situ hybridization (RNAscope) in Ago2 cKO compared to control germ cells. Data points show ratios for individual cells from at least three tubules. Bars show mean and 95% confidence interval. **p<0.01, Welch’s t-test. Sample images are shown in Figure S4C .

Article Snippet: Immunoblotting was performed using the following primary antibodies and dilutions: Histone H3 (Abcam, ab1791, 1:40000 or Abcam, ab18521, 1:1000), AGO2 (Abcam, ab186733, 1:2000 or Abcam, ab32381, 1:1000), H3K9me3 (Abcam, ab8898, 1:4000), HMGB2 (Abcam, ab124670, 1:3000), DAZL (Abcam, ab34139, 1: 2000), YBX2 (Abcam, ab154829, 1:4000), AK7 (Novus Biologicals, NBP2-92176, 1: 3000), GTSF1 (NOVUS Biologicals, NBP1-83934,1:1000), PTBP2 (Proteintech, 55186-1-AP, 1:4000), α-TUBULIN (Santa Cruz, sc-8035, 1:1000), SYCP3 (Abcam, ab97672, 1:1000), ACRV1 (guinea pig, gift of Dr. Prabhakara Reddi, University of Illinois , 1:5000).

Techniques: Alternative Splicing, Control, RNA In Situ Hybridization, RNAscope

Journal: eLife

Article Title: NMDA receptors in visual cortex are necessary for normal visuomotor integration and skill learning

doi: 10.7554/eLife.71476

Figure Lengend Snippet: ( A ) We injected an adeno-associated viral vector (AAV) to express Cre recombinase unilaterally and another to express a calcium indicator bilaterally (GCaMP6f) in V1 of ΔGrin1 mice. ( B ) Experimental timeline: a first group of Grin1 mice (ΔGrin1 juv ) was dark-reared from birth. We injected an AAV to express Cre at postnatal day (P)21 unilaterally in V1, injected a second AAV bilaterally to express GCaMP6f, and implanted imaging windows bilaterally at P30. A second group of Grin1 mice (ΔGrin1 adult ) was reared normally and received the same injections at p>100. All mice then had six sessions of visuomotor exposure in a closed-loop (CL) virtual environment before imaging experiments. ( C ) Example two-photon images showing co-expression of GCaMP6f and Cre-mCherry constructs. ( D ) In situ hybridization against Grin1 mRNA (see Materials and methods) confirming the local knockout of Grin1 in V1. Blue: hematoxylin stain for cell nuclei; brown: Grin1 hybridization signal. Brain regions were identified using a mouse brain atlas . ( E ) Injection sites of Cre were readily visible in Grin1 in situ hybridization images. Outside of the injection site (i), labeling was dense in most cells with multiple puncta per cell. In injection sites (ii), labeling was almost completely absent. Inset shows a cell with one punctum (arrow); if a cell had more than two of these puncta, it was counted positive in the analysis shown in ( F ). ( F ) The fraction of cells positive (more than two puncta per cell) for Grin1 mRNA in 0.5 mm × 0.5 mm regions in injection sites (Δ) was strongly reduced compared to regions outside of injection sites (C). ( G ) Grin1 knockout reduced expression of Grin1 in all major cortical neuron types. Left: violin plots of the number of single-cell sequencing mRNA reads corresponding to the portion of the Grin1 gene knocked out in the ΔGrin1 mice and control mice. Layer 2/3 (L2/3), layer 4 (L4), layer 5 (L5), and layer 6 (L6) excitatory neurons, and parvalbumin (PV)-positive, somatostatin (SST)-positive, and vasoactive peptide (VIP)-positive interneurons. Expression levels are normalized to the total number of reads per nuclei. Right: the same data for ΔGrin1 mice. Expression levels were significantly downregulated in all neuron types, with the exception of VIP neurons (see for statistics).

Article Snippet: For mapping viral expression, an mCherry sequence (Addgene 237633) was amended as a separate chromosome.

Techniques: Injection, Plasmid Preparation, Imaging, Expressing, Construct, In Situ Hybridization, Knock-Out, Staining, Hybridization, Labeling, Sequencing, Control