Journal:

Article Title: A Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus Pseudomonas (Sensu Stricto) in Environmental Samples

doi:

Figure Lengend Snippet: Primer mismatch testing in RDP with the CHECK_PROBE algorithm

Article Snippet: Selected environmental isolates from a variety of studies were identified with the Biolog microbial identification system according to the manufacturer’s recommendations. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Sequence source and taxonomic description a No. of primer/target mismatches b RDP ribotype description c Ps-for Ps-rev Beta (purple) bacteria Iodobacter fluviatile ATCC 33051 3 0 Neisseria group Burkholderia solanacearum ATCC 11696 1 0 Burkholderia subgroup I Burkholderia pickettii ATCC 27512 1 0 Burkholderia subgroup I Spirillum voluntans ATCC 19554 0 3 Spirillum voluntans group Azoarcus sp. strain S5b2 1 0 Rhodocyclus group Azoarcus indigens VB32 1 0 Rhodocyclus group Gamma (purple) bacteria Ectothiorhodospira halochloris ATCC 35916 >3 0 Ectothiorhodospira group Coxiella burnetii Q177 1 0 Legionella group Methylococcus capsulatus bath 2 0 Methylomonas group Oceanospirillum minutulum ATCC 19193 0 3 Halomonas group Moraxella osloensis 0 NS Pseudomonas and relatives group Moraxella osloensis SCB111 1 0 Pseudomonas and relatives group Pseudomonas aeruginosa ATCC 10145 0 2 Pseudomonas and relatives group d Pseudomonas aeruginosa ATCC 25330 1 0 Pseudomonas and relatives group d Pseudomonas aeruginosa NF13 0 NS Pseudomonas and relatives group d Flavobacterium lutescens ATCC 27951 0 0 Pseudomonas and relatives group d Pseudomonas mendocina ATCC 25411 0 0 Pseudomonas and relatives group d Pseudomonas flavescens B62 NCPPC 3063 0 0 Pseudomonas and relatives group d Pseudomonas putida PaW1 isolate mt-2 0 0 Pseudomonas and relatives group d Pseudomonas syringae A501 0 0 Pseudomonas and relatives group d Pseudomonas fluorescens ATCC 13525 0 0 Pseudomonas and relatives group d Azospirillum sp. strain DSM 1727 0 0 Pseudomonas and relatives group d Pseudomonas syringae 31R-1 0 0 Pseudomonas and relatives group d Pseudomonas syringae env.

Techniques: Bacteria

Journal: Journal of Functional Foods

Article Title: Membrane vesicles from the probiotic Nissle 1917 and gut resident Escherichia coli strains distinctly modulate human dendritic cells and subsequent T cell responses

doi: 10.1016/j.jff.2019.103495

Figure Lengend Snippet: Fig. 6. Quantification of the Th17 and Treg subsets induced by MV-stimulated DCs. (A-B) Flow cytometric analysis of CD4+ T cells co-incubated for 3 days with untreated mo-DCs (iDC) or mo-DCs primed with MVs of the indicated strains (10 μg/ml) at a DC:T cell ratio of 1:5 with specific markers for: (A) Th17 population (CD4+ CD196+) and (B) Treg (CD25+(High) FoxP3+) population. Representative dot plots of CD4+ cells co-incubated with iDCs (control) or with EcN MV- stimulated DCS are shown for each cell subset analysis. The percentage of positive cells is indicated in each quadrant or box. (C) Data (mean ± SEM) from three- independent experiments performed in triplicate are expressed as fold-change increase in the percentage of positive T cells for Th17 or Treg specific markers induced by MV-stimulated DCs with respect control iDCs induced values (set as 1, dot line). Statistical differences were evaluated by one-way ANOVA followed by Bonferroni’s test (normal distribution) *p < 0.05 versus control DCs.

Article Snippet: For surface marker staining, lymphocytes were first stained with anti-CD4 FITC (clone VIT4), anti-CD25 APC (clone 4E3) and anti-CD196 PE (clone REA190) mAb or with the corresponding isotype-matched conjugated irrelevant antibody (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Incubation, Control

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Generation and analysis of the Zic3 V5 and Zic3 ins5V mouse lines. (A) Schematic illustrating the site of CRISPR cleavage position relative to Zic3 and the homology between the donor template and genomic region, (A′) the desired V5 epitope tagged Zic3 mouse line ( Zic3 V5 ) and (A″) the arrangement of the insertion in the Zic3 ins5V mouse line. In A″, primers are shown as black half arrows and asterisk denotes the location of Kozak sequence. (B) The tail of the first Zic3 ins5V mouse and tail kinks are indicated with arrowheads. (C) PCR electrophoretogram displaying the amplicon produced by the insertion in Zic3 ins5V mouse using primers shown in A″. Note the amplicon is larger than the amplicons produced by either wild-type or Zic3 V5 /wt mice, indicating an undesired large insertion occurred. (D,E) Partial Sanger sequencing chromatogram of the PCR amplicons shown in C of a Zic3 ins5V/y mouse showing that (D) the right homology arm has been inserted into the 5′UTR and (E) the V5 tag was inserted within Zic3 exon 1. HA, homology arm; wt, wild-type; ntc, no template control.

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: CRISPR, Sequencing, Amplification, Produced, Control

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 ins5V mice display reduced viability

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques:

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Neural tube defects observed in Zic3 LacZ and Zic3 ins5V embryos. (A) Lateral view illustration of a 14.5 dpc embryo showing regions of the neural tube (adapted from ). (B-B′) Lateral and (B″) frontal views of a wild-type embryo. White lines in B indicate regions sectioned in F and H. (C) Lateral view of Zic3 LacZ/LacZ null embryo with a neural tube defect in the cervical region. (D) Lateral and (D′) frontal views of a Zic3 LacZ/y null embryo with a neural tube defect in the cephalic region. (E) Lateral and frontal (E′) views of a Zic3 LacZ/LacZ null embryo with midline facial defects. White lines in E indicate regions sectioned in G and I. (F) Section of wild-type embryo forebrain with two separate lateral/telencephalic ventricles and the third ventricle at the midline of the diencephalon. (G) Section of Zic3 LacZ/LacZ embryo forebrain displaying a single fused ventricle and lack of the third ventricle in the diencephalon. (H) Section of wild-type embryo head with two eyes on surface of the embryo. (I) Section of Zic3 LacZ/LacZ embryo head indicating one of the eyes is dysmorphic and located abnormally close to the midline. (J) Lateral view of Zic3 ins5V / ins5V embryo with a neural tube defect in the cervical region. (K) Lateral and (K′) frontal views of a Zic3 ins5V/y embryo with an encephalocele in the cephalic region. (L) Lateral and frontal (L′) views of a Zic3 ins5V/y embryo with a neural tube defect in the cephalic region. (M-M′) Lateral and (M″) frontal views of a Zic3 ins5V/y embryo with craniorachischisis and several facial defects. (N) Transverse section of embryo shown in (M) taken from the head at the level of the eye and stained with Hematoxylin and Eosin. Arrows point to lateral ventricles, arrowheads indicate eyes, and asterisk (*) denotes diencephalon. (B-E′, J-L′ and M-M′) scale bar: 2 mm; (F-I and M″) scale bar: 1 mm.

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Staining

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 ins5V embryos exhibit heart defects. (A-D) Gross embryonic hearts at 13.5-14.5 dpc and (A′-D″) transverse sections stained with Hematoxylin and Eosin. (A-A″) Wild-type heart with proper orientation of ventricles. (B-D) Heart looping defects found in Zic3 ins5V embryos, including (B) dextrocardia, (C) incomplete looping and (D) dextrocardia with incomplete looping. In addition to looping defects, Zic3 ins5V heart sections also revealed (B′-B″) a double outlet right ventricle (DORV) defect. (C′-C″) Atrial isomerism with an atrioventricular canal defect. (D′-D″) Atrioventricular canal defect and thin wall. Arrowheads point to atrioventricular canal defects and asterisks (*) denote atrial isomerism. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; PA, pulmonary artery; Ao, aorta. Scale bars: 500 µm.

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Staining

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 is overexpressed and undertranslated in Zic3 ins5V embryos. (A) Relative expression levels of Zic3 in wild-type ( n =3; wt, blue bar) and Zic3 ins5V ( n =5; red bar) embryos at the headfold stage (7.75 dpc) were assessed using Taqman probes for qPCR. Tbp was used for data normalization. Bars display geometric means and error bars represent geometric standard deviation (SD). Dots represent individual samples. (B) Whole-mount in situ hybridization using a riboprobe for Zic3 on 7.75 dpc wild-type ( n =13) and Zic3 ins5V ( n =9) embryos; scale bar: 250 µm. (C) Whole-mount in situ hybridization using a riboprobe for Zic3 on 10.5 dpc wild-type ( n =7) and Zic3 ins5V ( n =6) embryos; scale bar: 2000 µm. Purple staining in (B) headfold and primitive streak and (C) somites, limb buds and brain. A and P indicate anterior and posterior of embryos, respectively. (D) Western blot images for V5 (ZIC3) from nuclear enriched lysates of wild-type ( n =2), Zic3 V5 ( n =4) and Zic3 ins5V ( n =4) 10.5 dpc embryos. LAMIN B served as nuclear loading control. Full blots are shown in Fig. S3 .

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Expressing, Standard Deviation, In Situ Hybridization, Staining, Western Blot, Control

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 ins5V mRNA is predicted to contain a stem-loop hairpin structure. Predicted secondary structure of (A) Zic3 w t and (B) Zic3 ins5V via RNAfold. Scale indicates the base-pair probability, with red bases having the highest probability. The novel secondary structure is indicated by the thick arrow and the asterisk denotes the start codon location. (C) Schematic of hairpin generated in Zic3 plasmids. (D) Representative western blot image for FLAG (ZIC3) from NIH3T3 nuclear lysates. LAMIN B served as nuclear loading control. For each plasmid, n =3 independent transfections and western blots. -, untransfected; wt, wild-type mouse Zic3 (FLAG tagged) plasmid; 25 hp and 40 hp are mouse Zic3 plasmids with 25 bp and 40 bp stem hairpins, respectively. Full blots shown in Fig. S3 .

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Generated, Western Blot, Control, Plasmid Preparation, Transfection