Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Inhibition of Lloviu virus (LLOV) glycoprotein (GP)–driven entry by interferon-induced transmembrane (IFITM) proteins. A , 293T cells transduced to express human IFITM1, 2, 3 or chloramphenicol acetyltransferase (Cat) as a control were subsequently transduced with vectors bearing the indicated GPs and luciferase activity in cell lysates was determined. Means and standard errors of the mean (SEMs) are shown for 3 independent experiments performed with triplicate samples. Transduction of control cells was set as 100%. Abbreviations: FLUAV-HA, influenza A virus hemagglutinin; MLV-Env, murine leukemia virus . B , RD/ Gaussia luciferase (GLucC) cells transduced to express human IFITM 3 or Cat as control were spin-infected with VP40/N-terminal portion of GLuc virus-like particles (VLPs) bearing the indicated GPs, and the luciferase activity in cell lysates was determined. Means and SEMs are shown for 2 experiments conducted with quintuplicate samples. Infection of control cells was set as 100%. Similar results were obtained when 293T cells transfected to express IFITM3 were analyzed. C , Western blot analysis of IFITM expression in transduced 293T cells. The expression of wild type-IFITM3 and an IFITM3 mutant in which the amino acids SVKS were mutated to alanine was analyzed. D , Experiment was conducted as described for A , but the SVKS-AAAA mutant was analyzed. Means and SEMs are shown for 3 independent experiments. Transduction of control cells was set as 100%. Abbreviation: MARV, Marburg virus. E , Experiment was carried as described for B, but the IFITM3 SVKS-AAAA mutant was included, and VLPs bearing no GP served as negative control. Results of a single experiment conducted with quintuplicate samples are shown and were confirmed in a separate experiment; error bars indicate standard deviations.

Article Snippet: Sequences encoding previously described N- and C-terminal fragments of human codon-optimized GLuc [ ] were synthesized (Genscript).

Techniques: Inhibition, Virus, Control, Transduction, Luciferase, Activity Assay, Infection, Transfection, Western Blot, Expressing, Mutagenesis, Negative Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence

Journal: Scientific reports

Article Title: Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9.

doi: 10.1038/srep21918

Figure Lengend Snippet: Figure 1. Strategy for generating cellular and mouse models of chromosomal translocation via the ESC- and CRISPR/Cas9-based technologies. (a) Strategy for generating mESC models, or mESC-derived cellular models, and mouse models carrying a chromosomal translocation. (b) Strategy for generating site-specific chromosomal translocations in mESCs using the CRISPR/Cas9 system. Cdx2 and Gsk3α sgRNAs will guide Cas9 (blue) onto the indicated target sites located in mouse chromosome 5 (red) and chromosome 7 (green), respectively. DSBs will then be induced in these two sites. By activating NHEJ, DSBs can be repaired and the chromosomal translocation T (5:7) may occur in the designated location, thus generating two translocated chromosomes. To show the precise location and the relative length of the chromosomes, the chromosome graphs from the University of California Santa Cruz (UCSC) Genome Browser were used. Primer chr-short-p1 was designed to anneal to chromosome 7 at the site upstream of the predicted DSB point. Primer chr-short-p2 was designed to anneal downstream of the chromosome 5 DSB point. The size of PCR product is expected to be approximately 930 bp if the translocation occurs. Similarly, primers chr-long-p1 and chr-long-p2 were designed to detect T (5:7) chromosome-long, and the size of the PCR product is approximately 300 bp.

Article Snippet: Human codon optimized Cas9 gene was cloned from hCas9 plasmid (Addgene plasmid 41815) and inserted into the revised piggyBac expression vector with hygromycin resistance.

Techniques: Translocation Assay, CRISPR, Derivative Assay

Journal: Scientific reports

Article Title: Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9.

doi: 10.1038/srep21918

Figure Lengend Snippet: Figure 2. Translocation between chromosome 5 and chromosome 7 mediated by the CRISPR/Cas9. (a) PCR analysis with chr-short-p1 and chr-short-p2 primers showing the presence of a ~930 bp PCR product in E14-Cas9 mESCs infected with Cdx2 and Gsk3α -sgRNAs. (b) Sequence of the PCR product (in one pMD18-T clone) of the predicted T (5:7) chromosome-short, and one cytosine nucleotide was deleted at the junction point. (c) PCR analysis with chr-long-p1 and chr-long-p2 primers showing the presence of a ~300 bp PCR product in E14-Cas9 mESCs infected with Cdx2 and Gsk3α sgRNAs. (d) Sequencing of the PCR product (in one pMD18-T clone) of the predicted T (5:7) chromosome-long indicates the addition of five nucleotides at the junction point. (e) Fluorescent images of the metaphase chromosomes of mESCs labelled with chromosome 5 (red) and 7 (green) specific probes. Insets zoomed in the two translocated chromosomes. Scale bars represent 10 μ m.

Article Snippet: Human codon optimized Cas9 gene was cloned from hCas9 plasmid (Addgene plasmid 41815) and inserted into the revised piggyBac expression vector with hygromycin resistance.

Techniques: Translocation Assay, CRISPR, Infection, Sequencing